Enzyme delivery systems and methods of preparation and use

ABSTRACT

This invention relates to coated digestive enzyme preparations and enzyme delivery systems and pharmaceutical compositions comprising the preparations. This invention further relates to methods of preparation and use of the systems, pharmaceutical compositions and preparations to treat persons having ADD, ADHD, autism, cystic fibrosis and other behavioral and neurological disorders.

RELATED APPLICATIONS

This application is a divisional patent application of U.S. Utilityapplication Ser. No. 12/386,051, filed Apr. 13, 2009, and is alsorelated to international application no. PCT/US10/30895, filed Apr. 13,2010, which claims the benefit of U.S. Utility application Ser. No.12/386,051, each of which is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates generally to coated digestive/pancreaticenzyme preparations, and pharmaceutical compositions and enzyme deliverysystems comprising the preparations, as well as methods for theirpreparation, use, and controlled delivery in treating individuals withneurological or behavioral diseases or conditions susceptible totreatment with enzymes.

BACKGROUND

Digestive enzymes are produced by the salivary glands, glands in thestomach, the pancreas, and glands in the small intestines. For example,digestive enzymes produced by the pancreas and secreted into the stomachand small intestine aid in digestion. Digestive enzymes produced by thepancreas are secreted into the duodenum, or upper segment of the smallintestine, where the pH is around 5 to 6, and the enzymes assist in thedigestion of food components, including carbohydrates, lipids, proteinsand nucleic acids. However, when digestive enzymes are administeredorally, the enzymes are exposed to highly acidic conditions in thestomach, with a pH of around pH 1-2, as well as gastric proteases whichdenature and degrade the enzymes.

Digestive enzymes have been administered to mammals to treat enzymedeficiencies caused by conditions affecting the pancreas, such aspancreatitis and pancreatic enzyme deficiency. Pancreatic enzymesadministered to humans are commonly of porcine origin. Manufacturers ofenzyme preparations have also used enteric coatings for lipasecompositions in individuals with cystic fibrosis who requireadministration of lipases. The preparations for lipase delivery haveused enteric coatings containing, for example, hypromellose phthalate,dimethicone 1000, and dibutyl phthalate.

Certain methods for coating sensitive bioactive substances have beendescribed. U.S. Pat. No. 6,261,613 to Narayanaswamy et al. disclosesparticles that can contain yeast, coated in a shell of a fat in a betaprime form (i.e., triglyceride crystals having a blocky symmetry). Thecoating material can further contain emulsifiers such as those found inhydrogenated vegetable oil. However, the coating only allows release ofthe yeast in a limited temperature range of about 40° C. to about 55° C.U.S. Pat. No. 6,251,478 B1 to Pacifico et al. discloses certainsensitive substances including certain bioactive compounds encapsulatedin a lipid material.

No description in the Background section should be taken as an admissionthat such disclosure constitutes prior art to the instant invention.

SUMMARY OF THE INVENTION

The present invention relates to coated digestive enzyme preparations,and pharmaceutical compositions and enzyme delivery systems comprisingcoated digestive enzyme preparations, which are useful in the treatmentof individuals with autism, ADD, ADHD, Parkinson's disease, cysticfibrosis, other neurological and behavioral diseases or conditions. Thecoated and encapsulated digestive enzyme preparations of this inventionpermit controlled delivery of enzymes having increased stability andenhanced administration properties, to patients with neurological andbehavioral diseases and conditions susceptible to treatment withdigestive enzymes.

In some aspects, the present invention relates to a coated and/orencapsulated pancreatic/digestive enzyme preparation which comprises acore comprising digestive and/or 20 pancreatic enzymes and a coatingwhich comprises an emulsifiable lipid. The core contains an amount ofpancreatic/digestive enzyme effective for treatment of the patient'scondition, which can be, for example, a neurological disorder such asautism, ADD, ADHD, CF and Parkinson's disease, or other diseases forwhich an effective amount of pancreatic/digestive enzymes can beadministered. Among other properties, the coating protects thepancreatic/digestive enzyme from destabilizing factors such as solvents,heat, light, moisture and other environmental factors. The coating alsoprovides controlled release of the pancreatic/digestive when thecomposite is exposed to a solvent. In addition, in one aspect of thisinvention, the coated digestive enzyme preparations of this inventionhave improved pour properties, and improved taste and smell of thedigestive enzyme particles.

The invention also relates to a specific blend of enzymes and lipids forenzyme administration in individuals with Parkinson's disease, ADD,ADHD, autism and cystic fibrosis and other behavioral or neurologicalconditions and diseases. The coated digestive enzyme preparations can beused to obtain release at selected transit times or in selectedlocations of the gastrointestinal tract of humans. In one aspect, thisinvention relates to controlled release enzyme preparations.

In another aspect the invention relates to a coated digestive enzymepreparation comprising (a) a core containing a digestive enzymeparticle, where the enzyme present in an amount of from about 5% to 90%by weight of the particles; and (b) a coating comprising an emulsifiablelipid, wherein the coating continuously coats the core and theemulsifiable lipid emulsifies upon exposure to a solvent.

In another aspect, this invention relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of anencapsulated enzyme preparation, which comprises (a) a core whichcomprises an amount of pancreatic or digestive enzymes effective fortreating a subject suffering from autism, ADD, ADHD, Parkinson's'disease, cystic fibrosis, or other neurological condition or behavioraldisorder susceptible to treatment by the enzymes; and (b) a coatingcomprising an emulsifiable lipid.

In yet another aspect, this invention relates to an enzyme deliverysystem comprising encapsulated enzyme preparation having particles whichcomprise: (a) a core 20 comprising pancreatic or digestive enzymespresent in an amount of from about 5% to 95% by weight of the particles;and (b) a generally uniform coating to provide for controlled release ofthe enzymes, said coating comprising an emulsifiable lipid. In oneaspect, the encapsulated enzyme preparation particles of the enzymedelivery system are non-aerosolizable.

In certain aspects, the methods of preparing enzymes according to thisinvention produce coated enzyme preparations characterized, for example,by controlled rates of release, reduction in aerosolization and saferadministration, ability to be administered by a sprinkle/sachet deliverymethod, improved flow characteristics, enhanced shelf life and storagecapacity, and other properties described herein. In other aspects, thecoated enzyme preparation has improved pour properties which facilitatemanufacturing and packaging processes, for example packaging in pouchesand sachets.

In some aspects, the present invention is based on the surprising andunexpected discovery that certain coated digestive enzyme preparationswhich comprise a coating of emulsifiable lipid and a digestive enzymecore have favorable release and activity profiles and permit site timespecific and/or location specific targeted release along the GI tractfor the treatment of autism, ADD, ADHD, Parkinson's Disease and otherneurological or behavioral conditions susceptible to treatment withdigestive enzymes. In some aspects, the encapsulatedpancreatic/digestive enzyme preparations are prepared to obtain specificdelivery times or specific regions within the human gastrointestinal(GI) tract. In other aspects, the emulsifiable lipid composition ishydrogenated soy oil, but may be any suitable lipid or lipid blend.

The invention further relates in some aspects to more stable enzymepreparations protected against the environment to reduce, for example,degradation and/or denaturation of the enzymes. This permits delivery ofmore accurate doses of the enzyme preparation to treated individuals.The coating can also, in some aspects, provide emulsification when theenzyme preparations are contacted with appropriate solvents, while alsosurprisingly providing for controlled release of the enzyme in thegastrointestinal (GI) system. The emulsification properties of thecoating in a solvent allows for controlled release of the enzyme,preferably at selected locations in the GI tract, where enzymeutilization provides the most effective treatment.

The present invention also relates to methods of making the enzymepreparations by lipid coating and/or encapsulation of digestive enzymes.The methods comprise providing an emulsifiable lipid, and coatingscreened pancreatic/digestive enzyme particles with the lipid. Thedigestive enzymes comprise 5-95% of the coated enzyme preparations byweight.

In another aspect, as described herein, the inventors have surprisinglydiscovery that the methods of this invention can be used to producecoated digestive enzyme preparations comprising digestive and/orpancreatic enzymes coated with an emulsifiable lipid alone, or with alipid blend to achieve a controlled rate of enzyme release, withincreased release of the pancreatic/digestive enzyme upon exposure ofthe encapsulated preparation to a suitable solvent. The inventors havediscovered that encapsulated pancreatic/digestive enzyme preparationshaving a coating consisting essentially of one or more monoglyceridesexhibit increased release of the pancreatic/digestive enzyme uponexposure of the encapsulated composite to a solvent, such as water,while protecting against release in 0.1 N HCl.

The invention further relates to methods for administering the enzymepreparations. In some aspects, the methods include administering thepancreatic/digestive enzymes as coated preparations. In some aspects,the invention relates to a method of treatment comprising administeringto a subject with autism, ADD, ADHD, Parkinson's disease, cysticfibrosis, or other behavioral or neurological condition in need oftreatment with digestive enzymes, at least two doses of a compositioncomprising a therapeutically effective amount of an encapsulateddigestive enzyme preparation comprising a core comprising a digestiveenzyme; and a coating comprising an emulsifiable lipid. Determination ofwhether a subject is in need of treatment with an effective amount ofdigestive enzymes may be based on a determination that the subject hasan enzyme deficiency.

In addition, the invention relates to the delivery to humans ofpancreatic/digestive enzyme composites, preparations, enzyme deliverycompositions or systems comprising no or fewer excipients, carriers,additives and/or extenders, and/or requiring the use of no or fewersolvents' in the enzyme preparations. In some embodiments, the coatingconsists essentially of hydrogenated soy oil. This can reduce exposureto potentially toxic substances and will also reduce the possibility ofallergy formation. The invention further relates to the delivery ofpancreative and/or digestive enzymes with improved safety ofadministration.

In addition, the invention relates to methods of improved manufacturingresulting from the enhanced flow properties imparted to enzymepreparations by the lipid encapsulation. The lipid encapsulation ofpancreatic/digestive enzymes forms a lipid barrier to moisture whichpermits improved flow of the encapsulated enzyme preparations in thepackaging machinery.

The summary of the invention is not intended to be a complete orexhaustive recounting of every aspect of the invention described herein.Other aspects of the invention will be apparent from further descriptionset forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an electron micrograph of an unprocessed, raw digestiveenzyme particles;

FIG. 2 shows an electron micrograph of a coated enzyme preparationfollowing sieving and lipid coating of the raw digestive enzymepreparation;

FIG. 3 shows a bar graph particle size analysis for a raw digestiveenzyme particles with the % of particles that can pass through a USSSseive, as indicated on the y-axis;

FIG. 4 shows a bar graph of the % lipase activity in the raw digestiveenzyme particles, and following encapsulation, for coated enzymepreparations containing 70%, 80% and 90% digestive enzymes by weight;

FIG. 5 shows a bar graph of the % enzyme release for the enzymepreparations containing 70%, 80% and 90% digestive enzymes by weight, atthe times indicated on the x-axis;

FIG. 6 shows a bar graph of the particle size distributions of the rawdigestive enzyme particles compared with the particle size distributionsin coated enzyme preparations containing 70% or 80% digestive enzymes byweight;

FIG. 7 shows the flow chart for a process that can be used toencapsulate digestive enzyme particles;

FIG. 8 shows a chromatogram of peak area (mAU) vs. time for workingstandard (top line), diluent (line that starts third from the top whentime is 4 minutes), mobile phase used in the HPLC (bottom line at 4minutes) and placebo (second to the top line when time is 4 minutes),which demonstrate no interference with the standard trypsin peak.

FIG. 9 shows a graph of peak area (mAU) vs. sample concentration (mg/mL)for known trypsin concentrations obtained using HPLC to measure trypsinin the coated digestive enzyme preparation.

FIG. 10 shows fecal chymotrypsin (FCT) levels measured in nine childrenwith symptoms of autism.

FIG. 11 shows FCT levels measured in 26 children with symptoms ofautism.

FIG. 12 shows FCT levels measured in 46 children. 25 of the children hadsymptoms of autism, while 21 children did not have symptoms of autism.

FIG. 13 shows fecal chymotrypsin levels measured in 320 age-matchedchildren. The navy line (in grayscale, the upper, black line) shows FCTlevels for children with known conditions (genetic and otherconditions). The purple line (in grayscale, the upper, dark gray line),shows FCT levels for normal children without any known condition. Theaqua line, (in gray scale, the lower, medium gray line), shows FCTlevels for children with autism. The pink line (in gray scale, thelower, dark gray line), shows FCT measurements for children with ADHD.The yellow line (in grayscale, the lower, light gray line), shows FCTmeasurements for children with ADD.

FIG. 14 shows mean fecal chymotrypsin levels at baseline, and 30, 60, 90and 120 days after administration of Viokas or Ultrase enzymereplacement.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As described throughout, this invention relates in some embodiments tocoated digestive enzyme preparations, and pharmaceutical compositionsand enzyme delivery systems comprising coated digestive enzymepreparations, which are useful in the treatment of individuals withautism, ADD, ADHD, Parkinson's disease, cystic fibrosis, otherneurological and behavioral diseases or conditions.

Autism (sometimes called “classical autism”) is the most commoncondition in a group of developmental disorders known as the autismspectrum disorders (ASDs). Autism is characterized by impaired socialinteraction, problems with verbal and nonverbal communication, andunusual, repetitive, or severely limited activities and interests. OtherASDs include Asperger syndrome, Rett syndrome, childhood disintegrativedisorder, and pervasive developmental disorder not otherwise specified(usually referred to as PDD-NOS). It has been estimated that three tosix children out of every 1,000 will have autism.

Attention deficit-hyperactivity disorder (ADHD) is a neurobehavioraldisorder that affects 3-5 percent of all children in the US. Itinterferes with a person's ability to stay on a task and to exerciseage-appropriate inhibition (cognitive alone or both cognitive andbehavioral). Some of the warning signs of ADHD include failure to listento instructions, inability to organize oneself and school work,fidgeting with hands and feet, talking too much, leaving projects,chores and homework unfinished, and having trouble paying attention toand responding to details. There are several types of ADHD: apredominantly inattentive subtype, a predominantly hyperactive-impulsivesubtype, and a combined subtype. ADHD is usually diagnosed in childhood,although the condition can continue into the adult years.

Parkinson's disease (PD) belongs to a group of conditions called motorsystem disorders, which are associated with the loss ofdopamine-producing brain cells. The four primary symptoms of PD aretremor, or trembling in hands, arms, legs, jaw, and face; rigidity, orstiffness of the limbs and trunk; bradykinesia, or slowness of movement;and postural instability, or impaired balance and coordination. As thesesymptoms become more pronounced, patients may have difficulty walking,talking, or completing other simple tasks. PD usually affects peopleover the age of 50. Early symptoms of PD are subtle and occur gradually.In some people the disease progresses more quickly than in others. Asthe disease progresses, the shaking, or tremor, which affects themajority of PD patients may begin to interfere with daily activities.Other symptoms may include depression and other emotional changes;difficulty in swallowing, chewing, and speaking; urinary problems orconstipation; skin problems; and sleep disruptions.

Cystic fibrosis (CF) is one of the most common life-shortening, geneticdiseases. In the United States, 1 in 4,000 children are born with CF. Itis most common among western European populations; one in twenty-twopeople of Mediterranean descent are carriers of one gene for CF, makingit the most common genetic disease in these populations. CF is caused bya mutation in the gene, cystic fibrosis transmembrane conductanceregulator (CFTR). The product of this gene is a chloride ion channelimportant in creating sweat, digestive juices, and mucus. Although mostpeople without CF have two working copies (alleles) of the CFTR gene,only one is needed to prevent cystic fibrosis. Cystic fibrosis affectsthe exocrine (mucus) glands of the lungs, liver, pancreas, andintestines, causing progressive disability due to multisystem failure.CF can be characterized by, for example, 1) thick mucus production whichresults in frequent lung infections; 2) diminished secretion ofpancreatic enzymes causing poor growth, greasy stools, and deficiency infat-soluble vitamins; and 3) infertility in the males due to thecondition congenital bilateral absence of the vas deferens. Often,symptoms of CF appear in infancy and childhood. Meconium ileus is atypical finding in newborn babies with CF.

Enzyme preparations with non-lipid enteric coatings have been used todeliver lipases in individuals requiring administration of lipases toindividuals with cystic fibrosis in need of enzyme treatment. Inaddition, Fallon has described certain methods and enzyme compositionsfor use in treating children and other individuals, with autism, ADD,ADHD, Parkinson's disease and other neurological diseases or conditions,for example, U.S. Pat. Nos. 7,138,123, 6,660,831, 6,632,429; 6,534,063,hereby incorporated by reference as if set forth in full herein.

The nature of the human digestive tract creates challenges for thedelivery of digestive enzymes to patients with neurological andbehavioral conditions susceptible to treatment with digestive enzymes.Multiple temperature and pH changes over the course of the digestivetract make specific delivery a necessity and a challenge. For instance,pH as low as 1 is encountered in the stomach, but rapidly increases to amore basic pH of 5-6 in the proximal small intestine. For example,generally the pH in the stomach is approximately 1.2, the pH in theduodenum is about 5.0 to 6.0; the pH in the jejunum is about 6.8, andthe pH is about 7.2 in the proximal ileum and about 7.5 in the distalileum. The low pH in the stomach which changes rapidly to a more basicpH of 5-6 in the proximal small intestines, call for a specific delivery25 method depending upon where the enzyme is to be delivered.

For example, children with cystic fibrosis whose condition requiresadministration of lipases, require delivery of the lipases to the latterportion of the small intestine. In contrast, the inventors havedetermined that children with autism who need treatment with proteasesrequire delivery of those enzymes to the proximal small intestine.

Delivery of digestive enzymes can also be challenging due to the rapiddegradation and denaturing of enzymes at ambient room temperature, aswell as the enhanced degradation and denaturing that can occur with hightemperature, pressure, humidity and/or exposure to light. Moisture andheat together can quickly destabilize enzymes, reducing theireffectiveness, and shortening shelf life, leading to inaccurate dosing.Denaturization or destabilization of the enzymes can reduce theireffectiveness by reducing the dose of active enzymes to less than theamount needed for effective treatment. Alternatively, attempting tocompensate for the denaturization or destablization by increasing thedose to ensure an effective level of active enzyme, could risk anoverdose or overfilling a capsule or other dosage form. To protect andstabilize the pancreatic/digestive enzyme from unfavorable conditions,such a penetration, decomposition, the pancreatic/digestive enzyme(core) may be coated or encapsulated in a continuous coating containingan emulsifiable lipid. In another aspect, this invention provides newcoated enzyme preparations with improved shelf life.

Manufacturers of enzyme preparations have used enteric coatings todeliver lipases in individuals requiring administration of lipases, suchas individuals with cystic fibrosis. Because the porcine enzymes aredelivered in a mixture of proteases, lipases and amylases, and becausethese compositions for human consumption were prepared for lipasedelivery, the use of these enteric coatings, which include suchsubstances as hypromellose phthalate, dimethicone 1000, and dibutylphthalate, preclude delivery of proteases at the proper location in thedigestive tract. All other enzyme preparations presently on the marketcontain at least one of these enteric coating substances and/or otheradditives in the preparation. Some additives that enable manufacturing,such as additives to improve flow properties, may further risk patientreactivity or sensitivity to the enzyme preparation.

In one embodiment the present invention includes a coated digestiveenzyme preparation and/or composite, which, in some embodiments is anencapsulated pancreatic/digestive enzyme preparation. In other aspects,the invention includes enzyme delivery systems and pharmaceuticalcompositions comprising coated pancreatic/digestive enzyme preparations.These coated or encapsulated enzyme preparations contain corescomprising pancreatic or digestive enzyme particles, and a coatingcomprising an emulsifiable lipid.

The coatings in the digestive/pancreatic enzyme preparations create abarrier to degradation and denaturation, and allow more accurate levelsof active enzymes to reach the treated individuals. The lipid coating ofthis invention provides a significant barrier to moisture, heat,humidity and exposure to light by allowing for a physical barrier aswell as one that prevents and or reduces hydrolysis. The coated enzymepreparations undergo less hydrolysis as a result of protection frommoisture in the environment by the lipid coating. As a result of thepresent invention, pancreatic/digestive enzymes are provided which cantolerate storage conditions (e.g., moisture, heat, oxygen, etc.) forlong periods of time thus enabling extended shelf life. The coating ofthe encapsulated enzyme preparation protects the enzyme from theenvironment and provides emulsification in a solvent without detractingfrom the abrasion resistance of the coating. The invention thus furtherrelates to more stable enzyme preparations.

The coated enzyme preparations therefore reduce overfilling of theenzyme dosage, and enhance delivery of more accurate doses of the enzymeto individuals with autism, ADD, ADHD Parkinsons's disease, cysticfibrosis and other neurological or behavioral conditions or diseasessusceptible to treatment with pancreatic or digestive enzymes.

In addition, because children and other individuals with autism andother conditions often have multiple sensitivities to foods, additives,colorants and other carriers, excipients or substances used in drugformulations, it is a challenge to make an enzyme delivery system thatavoids the use of allergens, and other carriers, excipients, extenders,colorants, etc. that could potentially add to adverse symptoms or themorbidity of patients. Furthermore, in very young children an enzymedelivery system which allows ease and tolerability, is paramount. Asachet delivery system for these enzyme preparations has also heretoforenot been achieved.

It is another aspect of the present invention to make an enzymepreparation without the use of extenders colorants, dyes, flow enhancersand other additives to reduce the potential for allergens and othersensitivity reactions in children and other treated individuals. It hasbeen discovered that in some embodiments, the digestive enzymes cansurprisingly be encapsulated with a single lipid excipient to improveretention of enzyme activity, ease of administration, tolerability, andsafety of administration, among other properties. Surprisingly digestiveenzyme particles containing lipases can be successfully encapsulatedwith coating consisting essentially of only hydrogenated soy oil.

In addition, porcine pancreatic/digestive enzymes posses a significantodor and taste, similar to cured/smoked pork. This taste can be strongand offensive to some individuals taking enzyme replacement, andespecially to children. The addition of a lipid coating providessignificant taste masking to the enzyme preparation, which allows forthe tolerance of taste, as the lipid coating is odorless and tasteless.The use of this method of taste masking which does not involve the useof color, dyes, perfumes, recipients, or other substances is preferablefor the administration of medications, which have an unpleasant orundesirable taste and odor. In other embodiments, this invention relatesto coated digestive enzyme preparations with improved taste and smell.

In some embodiments, the coatings on the digestive enzyme particle coresare preferably continuous coatings. By “continuous,” it is meant thatthe pancreatic/digestive enzyme is uniformity protected. The continuouscoating of the fully surrounds or encapsulates the pancreatic/digestiveenzymes. The encapsulation provides protection of thepancreatic/digestive enzyme from conditions such as moisture,temperature, and conditions encountered during storage.

In addition, the encapsulation also provides controlled release of thepancreatic/digestive enzyme. The emulsification properties of thecoating in a solvent allows for controlled release of the enzyme in thegastrointestinal system, preferably the region of the GI tract where theenzymes are to be utilized. The coating of the encapsulated compositeprotects the enzyme from the environment and provides emulsification ina solvent without detracting from the abrasion resistance of thecoating. For example, for conditions requiring treatment with proteases,the release of the protease portion of the enzymes is necessary in theproximal small intestine, thereby necessitating a lipid encapsulationwhich has a dissolution profile between 30-90 minutes. The dissolutionprofile may also be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85or 90 minutes. Dissolution profiles may be obtained using methods andconditions known to those of skill in the art. For example, dissolutionprofiles can be determined at various pH's, including pH. 1, 2, 3, 4, 5,6, 7, 8, 9, 10.

The rate of release of the bioactive substance can also be controlled bythe addition of additives as described below. When the preparations areexposed to a solvent, the solvent interacts with the mollifiable lipidin the coating and results in emulsification of the coating and releaseof the bioactive substance.

“Encapsulate” as used herein means that the coating completely surroundsthe pancreatic/digestive enzyme. In a population of encapsulatedparticles, encapsulated enzyme preparations may include contaminating orsmall portion of particles with a substantially continuous coating aslong as the release profiles of the encapsulated particles are notsignificantly altered. A coated or encapsulated particle may contain oneor more digestive enzyme particles enveloped in one coating to form onecoated or encapsulated digestive enzyme particle in the coated orencapsulated digestive enzyme preparation.

The present invention also includes a method for preparing the enzymepreparations, pharmaceutical compositions, and delivery systems for thetreatment of neurological or behavioral disorders such as autism, ADD,ADHD Parkinsons's disease, cystic fibrosis and other behavioral orneurological conditions or diseases susceptible to treatment withpancreatic or digestive enzymes. By “susceptible to treatment withpancreatic or digestive enzymes” is meant that one or more symptoms ofthe disease or condition can be alleviated, treated, or reduced byadministration of an effective amount of pancreatic or digestiveenzymes.

In some aspects, the invention relates to the production of selectedcoated enzyme preparations made by coating digestive enzyme particleswith lipids not previously used in coated digestive enzyme preparations.The unique mixtures of emulsifiable lipids and enyzmes can delivercertain components of the pancreatic/digestive enzymes to selectedlocations and/or at selected times during transit of the GI tract. Insome aspects, the invention relates to methods of delivering digestiveenzymes to humans based upon dissolution profiles.

The emulsifiable lipid is any lipid, lipid mixture, or blend of lipidand emulsifiers which emulsifies when exposed to a solvent, and has amelting point which allows the lipid to be a solid at typical storagetemperatures. The emulsifiable lipid can be a vegetable or animalderived-lipid. In some embodiments, the emulsifiable lipid consistsessentially of, or comprises one or more monoglycerides, diglycerides ortriglycerides, or other components including, for example, emulsifiersfound in hydrogenated vegetable oils. In another embodiment the lipid isa non-polar lipid.

As used herein, animal and/or vegetable “derived” lipids can includefats and oils originating from plant or animal sources and/or tissues,and/or synthetically produced based on the structures of fats and oilsoriginating from plant or animal sources. Lipid material may be refined,extracted or purified by known chemical or mechanical processes. Certainfatty acids present in lipids, termed essential fatty acids, must bepresent in the mammalian diet. The lipid may, in some embodiments,comprise a Type I USP-National Formulary vegetable oil.

The digestive enzyme used in the present invention can be anycombination of digestive enzymes of a type produced by the pancreas,including, but not limited to digestive enzymes from a pancreatic sourceor other sources. The scope of the invention is not limited topancreatic enzymes of porcine origin, but can be of other animal orplant origin as well as those which are synthetically derived. Thedigestive enzyme may be derived from mammalian sources such asporcine-derived digestive enzymes. The enzyme may include one or moreenzymes, and can also be plant derived, synthetically derived,recombinantly produced in microbial, yeast, or mammalian cells, and caninclude a mixture of enzymes from one or more sources. Digestive enzyme,can include, for example, one or more enzymes from more or more sourcesmixed together. This includes, for example, the addition of singledigestive enzymes to digestive enzymes derived from pancreatic sourcesin order to provide appropriate levels of specific enzymes that providemore effective treatment for a selected disease or condition. One sourceof digestive enzymes can be obtained, for example, from ScientificProtein Laboratories (see Table 6). The digestive enzyme may be, forexample a pancreatin/pancrelipase composition. In one embodiment, thedigestive enzymes will comprise or consist essentially of 25 USPunits/mg protease, 2 USP Unit/mg, and 25 USP Units/mg amylase. The termdigestive enzyme may refer to one or more enzymes of a type produced bythe pancreas.

The digestive enzyme particles used as cores in the present inventioninclude digestive enzyme particles where about 90% of the particles arebetween about #40 and #140 USSS mesh in size, or between about 105 to425 μm, or where at least about 75% of the particles are between about#40 and #80 mesh, or about 180 to 425 μm in size. Particles between #40and #140 mesh in size pass through #40 mesh but do not pass through #140mesh. The coated or encapsulated digestive enzyme particles in oneembodiment of this invention may comprise less than about 35, 30, 25,20, 15 or 10% of the particles which can be sieved through #100 mesh(150 μm). In some embodiments, the term “non-aerosolizable” refers to acoated or encapsulated enzyme preparation where less than about 20% orless than about 15% of the particles can be sieved through #100 mesh(150 μm). The encapsulated digestive enzyme preparation can be anencapsulated digestive enzyme composite where the digestive enzymeparticles contain two or more enzymes.

The minimum amount of pancreatic enzyme present in the core is at leastabout 5% active enzymes by weight of the coated enzyme preparation, butin other embodiments may be at least about 30%, or at least about 50% byweight. The maximum amount of pancreatic/digestive enzyme present in thecomposite is at most about 95% by weight, and in other embodiments atmost about 90%, 85%, 80%, 75% or 70% of the coated enzyme preparation.In other embodiments, the amount of pancreatic enzyme present in thecomposite is about 10%, 15%, 20%, 25%, 35%, 40%, 45%, 55%, 60%, 65%,70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 87.5%, or 92.5% by weight oranywhere in between. At least about or at most about a of enzyme mayinclude equal to or about that % of enzyme. The term “about” includesequal to, and a range that takes into account experimental error in agiven measurement. As used in connection with particle sizes, the term“about” can refer to plus or minus 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% oranywhere inbetween. As used in connection with % particles that can besieved, the term “about” can refer to plus or minus 10, 9, 8, 7, 6, 5,4, 3, 2 or 1% or anywhere inbetween.

The composition which contains the encapsulated digestive enzymepreparation or composite can be delivered as a sprinkle, powder,capsule, tablet, pellet, caplet or other form. Packaging theencapsulated enzyme preparations in an enzyme delivery system thatfurther comprises single dose sachet-housed sprinkle preparations allowsfor ease of delivery, and accurate dosing of the enzyme, by allowing aspecific amount of enzyme to be delivered in each dosing. Allowing forspecific unit dosing of an enzyme preparation which maintains the enzymeactivity within specific stability parameters in an enhancement overother sprinkle formulations, which are housed, in a multi-unit dosingform that allows for air, moisture and heat to depredate and denaturethe enzyme preparation. In a preferred embodiment the powder or sachetis housed in a trilaminar foil pouch, or similar barrier to keep outmoisture and to protect the enzyme preparation from adverseenvironmental factors. The invention further relates to an improvementin stability due to a reduction in hydrolysis due to the lipidencapsulation.

Further the lipid encapsulation methodology reduces the aerosolizationof the enzyme preparation that may be caustic to the child if inhaledthrough the lungs or the nose. In another embodiment, the inventionincludes delivery of digestive enzymes with improved safety ofadministration, by reducing the amount of aerosolization of the enzyme.The lipid encapsulation reduces aerolization and the potential forcaustic burn, aspiration, and/or aspiration pneumonias in children andadministrators of the enzyme preparation, thereby reducing the potentialfor illness in already compromised children such as those with cysticfibrosis, and leading to safer administration.

As used herein, the term “non-aerosolizable” will be used to refer to acoated or encapsulated enzyme preparation where substantially all of theparticles are large enough to eliminate or reduce aerosolization uponpouring of the coated enzyme preparation compared to uncoated enzymeparticles. For example, the term “non-aerosolizable” may refer to acoated or encapsulated enzyme preparation where at least about 90% ofthe particles are between about #40 and #140 mesh in size, or betweenabout 106 to 425 μm, or where at least about 75% of the particles arebetween about #40 and #80 mesh, or about 180 to 425 μm. The term “nonaerosolizable” may also refer to a coated or encapsulated enzymepreparation where less than about 35, 30, 25, 20, 15 or 10% of theparticles can be sieved through #100 mesh (150 μm). In some embodiments,the term “non-aerosolizable” refers to a coated or encapsulated enzymepreparation where less than about 20% or less than about 15% of theparticles can be sieved through #100 mesh (150 μm).

As described and referred to herein, suitable pancreatic/digestiveenzymes and suitable coatings may be used in the compositions andmethods of this invention. The choice of suitable enzymes and ofsuitable lipid coatings, including choice of the type or amount ofenzymes or coating, are guided by the specific enzyme needs of theindividuals, and the selected diseases to be treated. The encapsulatedenzyme preparations that are one aspect of this invention have not beenpreviously described.

In some embodiments, the invention relates to specific blends of enzymesand lipids selected for delivery in individuals with Parkinson'sdisease, ADD, ADHD, autism, cystic fibrosis and other neurological andbehavioral disorders susceptible to treatment with digestive/pancreaticenzymes based on the transit times in the human gastrointestinal tract.It can further be based upon the need of the patient to be treated forvarious components of the digestive enzymes. Further, the inventionrelates to improvement of the delivery of digestive enzymes to humansbased specifically upon required delivery times, and dissolutionprofiles.

While general methods for coating certain sensitive biologic substanceshave been described, see, e.g., U.S. Pat. No. 6,251,478, herebyincorporated by reference, the encapsulated bioactive substance of thisinvention is an enzyme preparation comprising a core containingdigestive enzymes comprising or consisting of multiple proteases,lipases and amylases, and a coating which comprises or consistsessentially of an emulsifiable lipid.

Additives can be blended with the emulsifiable lipid. Selection of thelipid(s) and additives will control the rate of release of the bioactivesubstance. In the case of the digestive and or pancreatic enzymes, thelipid coat must be uniquely chosen to release the bioactive substance inthe area of the digestive tract selected for release to optimizetreatment.

The invention further relates to the administering of the coated and/orencapsulated enzyme preparation in a sachet or pouch preparation forease of delivery to children and adults. In some embodiments, theinvention specifically relates to the administration of a coated enzymeparticle preparation, housed in a sachet or pouch. This facilitatesadministration, including but not limited to, administration in food ordrink, direct administration into the oral cavity, or administrationdirectly into the GI system through an NG-tube, G-tube or other GIentrances or deliveries.

In some embodiments, each dose contains about 100 to 1500 mg of coatedor encapsulated enzyme preparation, and each dose may contain about 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400,1450, or 1500 mg of coated or encapsulated enzyme preparation. “About”can include 80 to 125% of the recited preparation. Each dose may also beplus or minus 10% of the recited weight. In one embodiment each doeswill have a protease activity of not less than about 156 USP units/mgplus or minus 10%. The protease activity may also be not less than about100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, or 200 USP units/mg.

In other embodiments, the invention relates to methods of treatmentcomprising administering to a subject with autism, ADD, ADHD,Parkinson's' disease, cystic fibrosis, or other behavioral orneurological condition susceptible to treatment with digestive enzymes,at least two doses of a composition comprising a therapeuticallyeffective amount of the coated digestive enzyme preparations. In certainembodiments, about 80% of the enzyme is released by about 30 minutes ina dissolution test performed at pH 6.0. In other embodiments, about 80%of the enzyme is released by about 30 minutes after the coated digestiveenzyme preparations reach the small intestine.

Another embodiment of the invention relates to the improvement ofdelivery of enzymes to humans by reducing the use of excipients,extenders and solvents currently used in the preparations for deliveryof digestive enzymes to humans. For example, the encapsulated digestiveenzyme preparation may contain only one excipient, which increases thesafety of administration by decreasing the chance of an allergicresponse. In one embodiment, the excipient is hydrogenated soy oil.

Because, in some embodiments, the lipid encapsulation method does notrequire the enzyme preparation to be treated with solvents, extendersand excipients to facilitate flow or improve stability, one aspect ofthe invention includes a “clean” preparation of GRAS substances(generally regarded as safe) to be administered. The reduction in theuse of solvents, extenders excipients and other additives permitted bythe methods of this invention reduces the exposure of the individualstaking the enzyme replacement to potential allergens, thereby producinga hypoallergenic enzyme preparation that further enhances its potentialuses in the treatment of individuals who might otherwise develop anallergic response to treatment. Administration of the coated enzymepreparations of this invention can thus reduce exposure to potentiallytoxic substances and will also reduce the possibility of allergyformation. Accordingly, in some embodiments, the encapsulated digestiveenzyme preparation is hypoallergenic.

The invention further relates in another aspect to the delivery ofdigestive enzymes with improved safety of administration. The lipid coatadds weight to the enzyme preparation, which reduces the potential foraerosolization. Previous uncoated enzymes have been shown to becomeaerosolized, and can therefore be inhaled and contact the nasal cavityor the lungs, causing injury to the mucosa of those taking and thoseadministering the enzyme preparation.

The invention further relates to the improvement of administering asachet preparation for delivery to children. The invention specificallyrelates to the administration of a coated digestive enzyme preparation,housed in a sachet which allows for particular types of administrationincluding but not limited to administration in food, drink, or directadministration into the oral cavity or directly into the GI systemthrough a NG-tube, G-tube or other GI entrances. The use of a sachetdelivery of enzymes has heretofore been not utilized in the enzymepreparations presently marketed. The sachet, which represents a unitdosage or multiple doses for a day, and represents a single unit dose.The sachet of a trilaminar foil allows the enzyme/lipid powder to remainstable, and allows for ease of administration.

In another embodiment, the invention relates to a method of controllingthe rate of release of the pancreatic/digestive enzyme from anencapsulated enzyme preparation upon exposure to a solvent. In someaspects, the method comprises blending an emulsifiable lipid with anamount of one or more additives to obtain a lipid blend; and coating thedigestive enzyme particle with the blend to form an encapsulateddigestive enzyme preparation containing particles comprising a corewhich contains the enzyme, and a coating which contains the lipid. Insome embodiments, the emulsifiable lipid is a blend where theemulsifiable lipid and additive are not the same, and where the rate ofrelease of the enzyme from the encapsulated composite upon exposure to asolvent is decreased as the amount of additive is increased. In thealternative, the rate of release of the enzyme from the encapsulatedcomposite upon exposure to a solvent is increased as the amount ofadditive is decreased.

The lipid coating surprisingly does not appear to be reduced ordestroyed by HCl (hydrochloric acid) present in the stomach, therebyprotecting the enzyme from degradation following administration untilthe enzyme preparation reaches its target region in the GI tract.Further the lipid coat reduces the exposure of the enzyme to attack bywater, thereby reducing hydrolysis, and further protecting the digestiveenzymes from degradation. In addition, the inventors have found that anexcipient containing only lipid can be used to coat or encapsulatedigestive enzyme particles containing lipase.

The use of digestive enzymes for the treatment of specific diseasetargets is made possible, in one aspect of the invention, by preparingencapsulated digestive enzyme composite having differing releasecharacteristics. Since various neurological and behavioral diseases canimpact the gastrointestinal systems in humans in various ways, the useof specific enzyme preparations and the ensuing encapsulation can makethe difference as to where and for what duration of time the enzymepreparation is delivered.

The invention therefore relates to improvement of the delivery ofdigestive enzymes to humans based specifically upon needed deliverytimes, and dissolution profiles. For example, in certain aspects of theinvention, the rate of release and dissolution characteristics areunique to the lipid encapsulations of this invention. The preparation ofcoated digestive enzymes using enzymes and lipids selected to optimizetreatment of behavioral and neurological conditions and diseasessusceptible to treatment with digestive enzymes has not been previouslydescribed.

As an example, previous enteric coatings for digestive and or pancreaticenzymes have delayed release of enzyme mixture for a period of time toolong for delivery of the protease portion to the proximal smallintestine. For instance, in administration to patients with cysticfibrosis where delivery of lipases is required for effective treatment,the dissolution profile of the enterically coated digestive enzymesneeds to favor a longer delay in the release of the enzymes, as well asthe delivery of a high lipase formulation.

Prior to the instant invention, lipid encapsulation had not been used asa delayed and/or protective mechanism for lipase delivery to treatindividuals with cystic fibrosis.

The inventors have further recognized that for treatment of patientswith autism who require delivery of protease enzymes for effectivetreatment, the lipid encapsulate can be modified to deliver the proteaseduring an earlier transit time window, in the proximal small intestine,to optimize protein digestion. In another example, the inventors haverecognized that for patients with Parkinson's disease who have slow GItransit times due to the dysautonomic nature of their neurologicalcondition, still another release profile is required to deliver enzymesfor effective treatment. The lipid and/or additive selection will bemade to obtain enzyme release at later times after administration.

It has not been previously appreciated that transit times for digestiveenzymes through the digestive system could be controlled by layeringlipids, or through encapsulation with specific lipid types. In stillanother aspect, this invention relates to a selected blend of enzymesand lipids for delivery in individuals with Parkinson's disease, ADD,ADHD autism and cystic fibrosis and other behavioral or neurologicaldiseases or conditions susceptible to treatment withpancreatic/digestive enzymes, based upon the transit times in thegastrointestinal systems of humans.

The invention further relates to an improvement in manufacturing due tothe enhanced flow properties imparted by the lipid encapsulation. Theimprovement in manufacturing can also accomplished through the lipidencapsulation of a pancreatic/digestive enzyme due to the lipid barrierto moisture thus allowing for improved flow in the packaging machinery.The improved flow qualities may facilitate packaging of the coateddigestive enzyme preparations into, for example, pouches or sachets.

In one aspect, this invention relates to the use of a lipidencapsulation method to make a coated digestive enzyme preparation forspecific delivery times within the human gastrointestinal (GI) tracttargeted for use in the treatment of a specific disease or condition.This disease or condition may be caused by or characterized by adigestive deficit that can be treated by the administration of digestiveenzymes to the appropriate region of the GI tract. The neurological orbehavioral disease or condition is one not traditionally associated withthe digestive system, where one or more symptoms can be treated byadministering an effective amount of a pancreatic and/or digestiveenzyme preparation.

Thus, the present specification is directed at lipid encapsulation ofspecific enzymes targeted for use in the treatment of specific diseases,and the encapsulation method includes the amount and type of lipids usedin the methods of this invention for the preparation of the encapsulateddigestive enzyme composite. The present invention also relates tomethods of making the enzyme preparations by lipid coating and/orencapsulation of pancreatic and/or digestive enzymes. The methodscomprise providing an emulsifiable lipid, and coatingpancreatic/digestive enzyme particles with the lipid, where thepancreatic/digestive enzymes comprise 5-90% of the coated enzymepreparations by weight. In some aspects the uncoatedpancreatic/digestive enzyme particles have a size range of about 105-425μm.

In one embodiment, the invention relates to a method of preparing anencapsulated digestive enzyme preparation, the method comprising a)screening uncoated digestive enzyme particles to obtain particles of asuitable size for encapsulation; and b) coating the screened digestiveenzyme particles with an emulsifiable lipid to form coated orencapsulated digestive enzymes containing a core which contains thepancreatic/digestive enzyme and a coating which contains theemulsifiable lipid. In some embodiments, the encapsulated digestiveenzyme preparation is a controlled release digestive enzyme preparation,which may have enhanced flow properties. The preparations may be usefulin the treatment of individuals with autism, ADD, ADHD, Parkinson's'Disease, Cystic fibrosis and other neurological conditions.

Screening of the particles may include quality control steps to improvethe activity, appearance or particle size of the digestive enzyme. Forexample, the particles may be analyzed to determine enzyme activitycontent, and/or visualized using chromatographic, microscopic or otheranalytical methods. The particles may also be screened to obtainparticles of a suitable size for encapsulation by removing particlesthat are too fine or too large. For example, the particles may be sievedto obtain particles of a suitable size or more uniform size range forencapsulation. As a further example, the particles may be sieved throughUSSS #40 mesh and through USSS #140 mesh. Particles that pass throughthe #40 mesh but are retained by the #140 mesh are of an appropriatesize range for coating or encapsulation Particles may also be screenedby sieving through USSS #140, #120, #100, #80, #70, #60, #50, #45, or#40 mesh, or any combination thereof.

Enzyme preparations supplied by the API supplier may be provided asirregular shaped, and multi-sized particles, with uneven edges, and muchclumping, and containing some crystalline salt particles. (See, forexample, FIG. 1). Uneven particle size and shape reduces flowproperties, and interferes with packaging. In addition, pouring uncoatedenzyme into the mouth of an individual would be difficult, andpotentially may cause too much or too little of the enzyme to bedelivered. Processing the digestive enzyme particles according tomethods in accordance with one aspect of this invention yields anon-dusty, free-flowing particulate preparation suitable for sachetpackaging and for pouring onto food or drink. In addition, as discussedthroughout, the use of lipid encapsulation to prevent aerosolization,and therefore increase safety, to increase flow properties which enhancemanufacturing of a pharmaceutical is an embodiment of the instantinvention.

The size distribution of particles in an exemplary raw enzymepreparation is shown in the graph in FIG. 3. Large particles (>40 mesh)and very small particles (<140 mesh) are generally not suitable forproper encapsulation and can be removed by screening. In order toincrease the flow properties of the encapsulated pancreatic enzymepreparation, digestive enzyme particles can be sieved to remove finesand overly large particles, for example by including only particles ofsizes 40-140 mesh, or about 105 to 425 microns. In some embodiments, thecoated digestive enzyme preparation containing 80% digestive enzyme byweight is made by coating sieved pancreatic enzyme particles with ahydrogenated vegetable oil using 20 lbs. of enzyme particles and 5 lbsof hydrogenated vegetable oil.

In some embodiments, the temperature of the lipid or lipid blend ismaintained at 110° F. before application to the digestive enzymes, whichare not heated.

In some embodiments, the lipid should be present in the preparation at aminimum amount of about 5% by weight of the encapsulated composite,preferably about 30%, and more preferably about 50% by weight of theencapsulated composite. The maximum amount of pancreatic/digestiveenzyme present in the encapsulated composite is about 95% by weight ofthe composite, preferably about 90%, and more preferably about 85% ofthe encapsulated composite. The emulsifiable lipid can be any lipid orlipid-derived material that emulsifies or creates an emulsion yet has amelting point which allows the emulsifiable lipid to be a solid attypical storage temperatures, for example, 23 degrees Centigrade.

“Emulsifiable lipids” as used herein means those lipids which contain atleast one hydrophilic group and at least one hydrophobic group, and havea structure capable of forming a hydrophilic and hydrophobic interface.These chemical and/or physical properties, mentioned above, of anemulsifiable lipid permit emulsification. Examples of interfacesinclude, for example, micelles and bilayers. The hydrophilic group canbe a polar group and can be charged or uncharged.

The emulsifiable lipid can be derived from animal or vegetable origins,such as, for example, palm kernel oil, soybean oil, cottonseed oil,canola oil, and poultry fat, including hydrogenated type I vegetableoils. In some embodiments, the lipid is hydrogenated. The lipid can alsobe saturated or partially saturated. Examples of emulsifiable lipidsinclude, but are not limited to, monoglycerides, diglycerides, fattyacids, esters of fatty acids, phospholipids, salts thereof, andcombinations thereof.

The emulsifiable lipid is preferably a food grade emulsifiable lipid.Some examples of food grade emulsifiable lipids include sorbitanmonostearates, sorbitan tristearates, calcium stearoyl lactylates, andcalcium stearoyl lactylates. Examples of food grade fatty acid esterswhich are emulsifiable lipids include acetic acid esters of mono- anddiglycerides, citric acid esters of mono- and di-glycerides, lactic acidesters of mono- and di-gylcerides, polyglycerol esters of fatty acids,propylene glycol esters of fatty acids, and diacetyl tartaric acidesters of mono- and diglycerides. Lipids can include, for example,hydrogenated soy oil.

Any emulsifiable lipid may be used in the methods and products of thisinvention. In certain embodiments the emulsifiable lipid used willproduce non-agglomerating, non-aerosolizing enzyme preparationparticles.

In other embodiments, the method relates to preparation of anencapsulated, controlled release digestive enzyme preparation withenhanced flow properties useful in the treatment of individuals withautism, ADD, ADHD, Parkinson's' Disease, Cystic fibrosis and otherneurological conditions, the method comprising: a) blending anemulsifiable lipid with one or more additives to obtain a blend; and b)coating screened digestive enzyme with the blend to form an encapsulateddigestive enzyme containing a core which contains the digestive enzymeand a coating which contains the blend of emulsifiable lipid.

The coating of the enzyme with the lipid, as shown in FIG. 2, allows forthe enzyme to become more uniform in size and shape, but reduces thejagged edges associated with the raw enzyme, and allows for ease ofadministration and ease of manufacturing, as the flow propertiesassociated with the covered enzyme will allow for the manufacturingmachinery to easily fill the sachet/pouch with the enzyme and reducesoverfilling or under filing of the sachet. The unit dose packagingreduces the ability of the child to open the multi dose can/box/or othercontainer. The trilaminar foil pouch or sachet further reduces theability of a child to open the sachet/pouch, and over utilize theenzyme.

In another embodiment, the invention relates to a method of controllingthe rate of release of a digestive enzyme from the encapsulatedpreparation by using a lipid blend to coat the digestive enzyme. Themethod includes blending an emulsifiable lipid with one or moreadditives to obtain a blend, and coating the digestive enzyme with theblend to form an encapsulated digestive enzyme containing a core whichcontains the digestive enzyme and a coating which contains the blend ofemulsifiable lipid. The rate of release of the enzyme from theencapsulated preparation upon exposure with a solvent is decreased asthe amount of additive is increased. In the alternative, the rate ofrelease of the enzyme from the encapsulated composite upon exposure witha solvent is increased as the amount of additive is decreased. Thus, thenature of the coating allows for controlled release of the enzyme fromthe encapsulate.

Non-emulsifiable lipids do not possess the chemical and/or physicalproperties related to emulsification as described above and include anylipid, lipid derived material, waxes, organic esters, or combinationsthereof. Non-emulsifiable lipids generally do not emulsify bythemselves. Non-emulsifiable lipids can be used as additives so long asthe properties of the coating, and constituent lipids, permitemulsification. Non-emulsifiable lipids, such as, for example,triglycerides, can be blended with an emulsifiable lipid of the presentinvention. The non-emulsifiable lipid can be derived from animals,vegetables, mineral, or synthetic origins. The non-emulsifiable lipid ispreferably hydrogenated, and can be saturated or partially saturated,and includes, but is not limited to triglycerides. In a preferredembodiment, the coating contains a blend of monoglycerides andtriglycerides applied to a pancreatic/digestive enzyme.

The inclusion of one or more additives with an emulsifiable lipid of thepresent invention is used to control emulsification of the coating andrelease of the enzyme. For example, the additive, triglyceride, can beblended with monoglycerides (e.g., an emulsifiable lipid), to controlemulsification of the coating and thus control (e.g., decrease) the rateof release of the enzyme from the composite. As a further example, oneor more additives, such as a diglyceride and a triglyceride can beblended with the emulsifiable lipid to control the rate of release ofthe enzyme. Hydrogenated vegetable oils may contain emulsifying agents,such as soy lecithin or other components.

Properties including mechanical strength, melting point, andhydrophobicity can be considered when choosing a suitable lipid coatingfor the digestive enzyme. Lipids having lower melting points or morepolar, hydrophilic properties were generally less suitable forencapsulation because they resulted in product that would cake underaccelerated storage stability conditions. Enzyme preparations madeusing, for example, hydrogenated soy oil, hydrogenated castor wax, andcarnauba wax all demonstrated good pouring and no caking.

The wax can be paraffin wax; a petroleum wax; a mineral wax such asozokerite, ceresin, or montan wax; a vegetable wax such as, for example,carnuba wax, bayberry wax or flax wax; an animal wax such as, forexample, spermaceti; or an insect wax such as beeswax.

Additionally, the wax material can be an ester of a fatty acid having 12to 31 carbon atoms and a fatty alcohol having 12 to 31 carbon atoms, theester having from a carbon atom content of from 24 to 62, or a mixturethereof. Examples include myricyl palmitate, cetyl palmitate, myricylcerotate, cetyl myristate, ceryl palmitate, ceryl certate, myricylmelissate, stearyl palmitate, stearyl myristate, and lauryl laurate.

In a further embodiment, the invention provides a method for controllingrate of release of a pancreatic/digestive enzyme from an encapsulatedcomposite upon exposure to a solvent. The method includes coating theenzyme with an amount of an emulsifiable lipid to form an encapsulatedpancreatic enzyme substance composite, wherein the rate of release ofthe enzyme from the encapsulated composite is decreased as the amount ofemulsifiable lipid based on total weight of the encapsulated compositeis increased. In the alternative, the rate of release of the pancreaticenzyme from the encapsulated composite is increased as the amount ofemulsifiable lipid based on total weight of the encapsulated compositeis decreased. The emulsifiable lipid useful in this embodiment canconsists essentially of one or more monoglycerides.

The solvent in which a lipid emulsifies can be an aqueous solvent. Theaqueous solvent interacts with the hydrophilic groups present in theemulsifiable lipid and disrupts the continuity of the coating, resultingin an emulsion between the aqueous solvent and the lipids in thecoating, thus releasing the bioactive substance from the composites.

The methods herein, used to encapsulate pancreatic or digestive enzymecores for treatment of neurological conditions or disorders, has notbeen previously described. The methods for lipid encapsulation ofmedications for human consumption which have the characteristics of atime-released medication, and which utilize the lipid encapsulation forstability have not been previously described. Prior to the experimentsdescribed herein, there was no published protocol that allowed for thepreparation of an encapsulated enzyme preparation comprising a coatingof emulsifiable lipid and a digestive enzyme suitable for thetime-specific arid/or site-specific targeted release along the GI tractfor the treatment of autism, ADD, ADHD, Parkinson's Disease and otherneurological or behavioral conditions susceptible to treatment withdigestive enzymes.

Aspects and embodiments of the instant disclosure stem from thesurprising and unexpected discovery that certain pharmaceutical dosagepreparations comprising a coating of emulsifiable lipid and a digestiveenzyme can have novel potentiated activity and unexpected favorablerelease and dissolution profiles and absorption kinetic parameters alongthe various portion of the GI tract. These characteristics are usefulfor formulating a specific bioactive enzyme for site specific targetedrelease along the GI tract for the treatment of autism, ADD, ADHD,Parkinson's Disease and other neurological conditions.

Determination of whether a subject is in need of treatment with aneffective amount of digestive enzymes may be based on a determinationthat the subject has an enzyme deficiency.

In one aspect of the invention, the method comprises using the enzymeformulations of this invention to treat children and other individualswith autism, ADD, ADHD, Parkinson's disease and other neurologicaldiseases or conditions, who also have an enzyme deficiency. The enzymedeficiency could be determined by any method used in determining ordiagnosing an enzyme deficiency. In one aspect the determination ordiagnosis may be made by evaluating symptoms, including eating habits,self-imposed dietary restrictions, symptoms of eating disorders and/orgastrointestinal disorders. In other aspects, the determination may bemade on the basis of a biochemical test to detect, for example, levelsor activities of enzymes secreted, excreted or present in the GI tract,and/or by determining the presence of a mutation in a gene or aberrantexpression of a gene encoding one or more digestive enzymes. The enzymedeficiency may also be determined, for example, by detecting a mutationor aberrant expression of a gene encoding a product regulating orotherwise affecting expression or activity of one or more digestiveenzymes.

In some aspects, the individual to be treated may also be tested for thepresence of a co-morbidity, which is a co-morbidity which does notaffect the activity or expression of a digestive enzyme. In certainaspects, individuals who are determined to have autism based on clinicalsymptoms but not a co-morbidity such as a genetic co-morbidity, aretreated with the enzyme delivery systems described herein. However,individuals who are determined to have autism based on clinical symptomsand a co-morbidity, who nevertheless also test abnormally low for FCTlevel or positive using another indicator of GI pathogens and/or lowdigestive enzyme activity or expression may also be treated with theenzyme delivery systems of this invention.

The following co-morbidities are set forth as exemplary co-morbidities:

Fragile X

Hallermann-Streiff syndrome Trisomy 21

tranlocation on 9

Beckwith-Wiedemann syndrome

Trisomy 21

Trisomy 18

Rubenstein-Tabi syndrome

Fragile X

Prader-Willi syndrome

Trisomy 21

Rett syndrome

Klippel-Feil syndrome

Rett syndrome

Duchenne Muscular Dystrophy

Tourette syndrome

In-utero stroke Trisomy 21

Fragile X

Juvenile RA

In-utero stroke

Trisomy 6

Duchenne Muscular Dystrophy

Juvenile Diabetes

Diabetes Type I

Adrenoleukodystrophy

Wilson's disease

In-utero stroke

Diabetes Type I

Prader-Willi syndrome

22q13

Tourette syndrome

Lissencephaly

Neutrophil Immunodeficiency syndrome

Diabetes Type I

Tourette syndrome

Tetrasomy 18p

Hyper IgE syndrome

Angelman Syndrome

Diabetes Type I

Rett syndrome

Fragile X

Marfan syndrome

Waardenburg syndrome

glutathione synthetase deficiency

Diabetes Type I

Rubinstein-Taybi

Angelman Syndrome

Klinefelter Syndrome

Brain bleed at birth

Turner Syndrome

Hypothyroidism

Diabetes Type I

Brain damage of prematurity

In One aspect, the determination of an enzyme deficiency may be madeusing a test for fecal chymotrypsin levels. Methods such as PCR or otheramplification, SNP detection, sequencing, and/or DNA combing may be usedto detect the presence of a mutation or presence of short RNA sequenceswhich interfere with expression of one or more genes encoding adigestive enzyme. For example, the mutation may in a gene encoding adigestive enzyme which decreases or eliminates the activity of theenzyme. As another example, the mutation may be mutation in the METgene, a gene encoding the pleiotropic MET receptor tyrosine kinase SeeCampbell et al., PNAS 103(46), 16834-39 (2006). These mutations mayinclude, for example, the MET promoter variant rs1858830 C allele, andor mutations in the MET signaling pathway such as a haplotype of theSERPINE1 gene, or the rs 344781 PLAUR promoter variant T allele.

The enzyme formulations of this invention are suited for use indelivering digestive enzymes to individuals with autism, ADD, ADHD,Parkinson's disease and other neurological diseases or conditions inneed of enzyme treatment. Fallon has described certain methods andenzyme compositions for use in treating children and other individuals,with autism, ADD, ADHD, Parkinson's disease and other neurologicaldiseases or conditions, for example, U.S. Pat. Nos. 7,138,123,6,660,831, 6,632,429, 6,534,063, hereby incorporated by reference as ifset forth in full herein.

The present invention will now be described more fully with reference tothe accompanying figures and examples, which are intended to be read inconjunction with both this summary, the detailed description, and anypreferred and/or particular embodiments specifically discussed orotherwise disclosed. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided byway of illustration only and so that this disclosure will be thorough,complete, and will fully convey the full scope of the invention to thoseskilled in the art.

In the experiments described herein, several factors were discoveredthat allowed for the unexpected enhanced/potentiated efficacy andproperty. For example, it was discovered that certain encapsulationenzymatic preparations comprising soy oil exhibited certain surprisingcharacteristics that led to improvements in the site-specific activity,release/dissolution profile, and ease of manufacturing, packaging andstorage. Without being bound to a particular theory of operation, theskilled artisans will appreciate that other methods of samplepreparation and/or formulation that can also yield these advantageousparameters are also contemplated herein.

The following experiments describe exemplary procedures in accordancewith the invention. It is to be understood that these experiments andcorresponding results are set forth by way of illustration only, andnothing therein shall be construed as a limitation on the overall scopeof the invention. By way of example, these studies demonstrate some ofthe unexpected improvements realized by the exemplary encapsulatedenzyme preparations of the present disclosure.

Example 1 Increased Flow Properties and Pourability of an ExemplaryEncapsulated Digestive Enzyme Preparation

Before the exemplary methods and preparations of the present disclosureis applied, examination of an unprocessed, raw enzyme preparation(Scientific Protein Laboratories (SPL) of Wanakee, Wis.) revealed thatit contained significant variability in particle size and 20 irregularmorphology, as shown in an electron micrograph of the particles aspictured in FIG. 1. Some crystalline salt particles are also visible.The raw enzyme does not pour as it clumps and is difficult to measuredue to the uneven surfaces, and jagged edges. The raw preparation isalso not suitable for lipid encapsulation without further processingbecause the raw product contains particles both too large and too smallfor proper encapsulation. The sieved enzyme, while more uniform in size,continues to exhibit uneven surfaces and clumps while pouring.

FIG. 2 shows the coated enzyme preparation produced following sievingand lipid coating of the raw material. In this example, the morphologyof particles is significantly improved, with rounder surfaces. Thisleads to a non-dusty product with good flow and organoleptic properties.

The morphology of the enzyme is now greatly improved due to the roundingof the surfaces, which leads to a product which is less dusty, does notaerosolize and has good flow and improved organoleptic properties.

The size distribution of particles in the raw enzyme preparation isshown in the graph in FIG. 3. In general, large particles (>40 mesh) andvery small particles (<140 mesh) are not suitable for properencapsulation. In order to increase the flow properties of theencapsulated pancreatic enzyme preparation, the raw enzyme particleswere sieved to include only particles of sizes 40-140 mesh, or about 106to 425 microns.

Example 2 Stability of an Exemplary Encapsulated Digestive EnzymePreparation: Temperature Storage

In a further exemplary embodiment, multiple types and weight percentagesof lipids were used to coat the sieved enzyme cores. Propertiesincluding mechanical strength, melting point, and hydrophobicity weretaken into consideration in choosing a suitable lipid coating for thepancreatic enzyme. Multiple examples of lipid coatings were examinedbelow and their physical appearances were examined under 25° C. and at40° C. Accordingly, lipids with a range of physical properties such asmechanical strength, melting point and hydrophobicity were evaluated forcoating of the pancreatic enzymes. In this example, it was found thatthe decreasing the melting point or increasing the hydrophilicity of thecoatings were not suitable for encapsulation because they resulted inproduct that would cake under accelerated storage stability conditions.The sieved and encapsulated enzyme preparations made using hydrogenatedsoy oil, hydrogenated castor wax, and carnauba wax all demonstrated goodpouring and no caking.

Table 1 provides the results of the visible physical changes whichoccurred at 25° C. and 40° C.:

Physical Appearance Physical Appearance at Coating System at 25° C.storage 40° C. storage Hydrogenated Soy oil OK OK (Balchem/Alibec)Hydrogenated Castor OK OK wax Carnauba Wax OK OK Hydrogenated OK SomeCaking Monoglycerides Soy/Monoglcyeride OK Some caking blends

Both the hydrogenated monoglycerides and the soy oil/monoglycerideblends demonstrated caking at the higher temperature. Therefore it isclear that the lower melting or more hydrophilic coatings were notsuitable for encapsulation because they resulted in a product that wouldcake under extended storage conditions as evidenced by our acceleratedstorage condition test at 40 degrees Centigrade.

Both the hydrogenated monoglycerides and the soy oil/monoglycerideblends demonstrated caking at the higher temperature. Therefore it isclear that decreasing the melting point or increasing the hydrophilicityof the coatings were not suitable for encapsulation because theyresulted in a product that would cake under extended storage conditionsas evidenced by our accelerated storage condition test at 40 degreesCentigrade.

Example 3 An Exemplary Encapsulated Digestive Enzyme PreparationSuitable for Pancreatic Enzymes: Enzyme Activity Measured as a Functionof Stability

In a further embodiment, enzyme stability was determine according to thefollowing method: For the accelerated test, standard ICH guidelines wereused: the coated preparations were placed in a plastic container, whichwas stored in a controlled humidity cabinet at 40° C. and 75% relativehumidity. Enzymatic activity was measured by grinding the coated enzymepreparations, dispersing in appropriate buffers, and testing for lipaseactivity.

TABLE 2 PERCENT STABILITY OF ENCAPSULATED ENZYMES WHEN STORED AT 40C/75% RH, IN CLOSED CONTAINERS Activity Activity Activity Activity 1week 2 weeks 1 Month Sample Lot# or coat RT Capped Capped capped PEC rawNov ′06 1206-1369A 116% 126% 75% PEC encap 70%, R1C-0890 118% 112%monoglyceride PEC encap 50%, R1C-0891 116% 110% 88% soy/mono PEC raw Jan′07 1206-1382B 113% 61% PEC encap R1C-0898 70% carnuba PEC encapR1C-0898 68% 50% carnuba PEC encap castorwax 108% 78% 87% 70%, castorwax PEC encap soy 99% 89% 87% 80%, soy

As illustrated above in the data summarized in Table 2, the soy oil 80%appeared to impart the greatest amount of stability of all the lipids,an effect that surprisingly was greater for enzyme preparations storedin capped containers than in uncapped containers. Tests of stability for75% relative humidity enzyme preparations stored at 40° C. in open pansdid not show significant differences in stability between coated anduncoated preparations.

Example 4 An Exemplary Encapsulated Digestive Enzyme PreparationSuitable for Pancreatic Enzymes: Enzyme Activity and Rate of Release ofMultiple Soy Encapped Pancreatic Enzyme

In a further embodiment, encapsulates were prepared according to themethods described below. The raw enzyme material was sieved to obtainparticles smaller than 40 mesh but larger than 140 mesh, to removefines, and to obtain a more uniform mixture more suitable for entericcoating.

The following preparations were made:

70% active enzyme by weight, with a standard stable soy coating;

80% active enzyme by weight, with a standard stable soy coating; and

90% active enzyme by weight, with a standard stable soy coating.

Activity in each encapsulated enzyme preparation was measured bygrinding the encapsulates, dispersing the ground material in appropriatebuffers, and testing for lipase activity.

As shown in FIG. 4, the enzyme activity in the coated preparations doesnot show any significant loss of activity upon coating (decrease from110 to 100% activity, normalized to stated enzyme activity of the rawenzyme material).

Enzyme release was measured by suspending each encapsulate in adissolution apparatus at pH 6.0 buffer for 30, 60, and 90 minutes (100rpm, as per USP guidelines). As shown in FIG. 5, all encapsulates showbetween 80-90% release at 30 and 60 minutes. At 90 minutes, the measuredenzyme activity obtained with these preparations decreases.

Example 5 An Exemplary Encapsulated Digestive Enzyme PreparationSuitable for Pancreatic Enzymes: Particle Size of Multiple Soy OilEncapped Pancreatic Enzyme

In a further embodiment, preparations containing 70% or 80% activepancreatic enzyme by weight, encapsulated with soy oil were compared toraw pancreatic enzyme material with respect to particle size, as shownin FIG. 6.

All levels of lipid demonstrate an impact of particle size. The 80% PECdemonstrates the most uniform as none appear at the 200 mesh level.

Example 6 An Exemplary Encapsulated Digestive Enzyme PreparationSuitable for Pancreatic Enzymes: Smell and Taste

Examination of exemplary encapsulated enzyme preparations containing70%, 80% and 90% enzyme by weight were performed to determine theirtaste and smell when compared to Sucanat™ and brown sugar, as well ascompared to the raw enzyme. The results are shown in Table 4, below.Sucanat™ is an organic whole food sweetener.

TABLE 4 SUBSTANCE ODOR TASTE Brown sugar Yes Sweet Sucanat ™ No SweetRaw Enzyme Meaty/smoky N/A 70% No No 80% No No 90% Slight Salty

Example 7 An Exemplary Encapsulated Digestive Enzyme PreparationSuitable for Pancreatic Enzymes: Manufacturing

The flow chart outlining the manufacturing process useful in making theenzyme preparations of this invention is shown in FIG. 7.

Ingredients used in making a batch of an exemplary encapsulatedpancreatic enzyme preparation included 20.0 lb. of sieved pancreaticenzyme and 5.0 lb. of hydrogenated vegetable oil, for example, soy oil.

The pancreatic enzyme concentrate was first sieved through a 40 USSSmesh screen, and the material which passed through the mesh wasretained. The retained material was then screened through a 140 USSSmesh screen (or the equivalent), and the material which did not passthrough the mesh was retained as the sieved pancreatic enzyme materialor particles.

In the encapsulation process, the appropriate coating material ischarged to the melt pot, and brought to and maintained at 110° F. forthe spraying process. Any temperature that will provide appropriateconsistency during the spraying process may be used. In someembodiments, the temperature is further selected based on the meltingpoints of the lipids used in the coating, and/or so that after contactof the sieved pancreatic enzyme material or particles with the coating,the activity of the enzyme preparation remains about the same.

The liquefied coating material is weighed and transferred to the spraypot. The sieved pancreatic enzyme was added to the encapsulationmanufacture vessel. The pancreatic enzyme particles are encapsulatedwith coating material to the selected coating level.

The encapsulated material is screened with a 14 USSS mesh screen (orequivalent), and the material that passes through the screen isretained. Following sieving, the material is collected and samples areremoved for QC.

If two sub-batches are to be blended, the loaded screened material isadded to a suitable blender and blended for 7 to 10 minutes. Samples areobtained for finished product testing. The encapsulated material is bulkpackaged and placed in quarantine pending test results. Upon achievingacceptance criteria, the finished product is released by the Qualitygroup. Afterwards, the product may be shipped as directed.

Samples are collected for finished product testing, including analyticaltesting and microbial assays, which can be tested over time.

Example 8 An Exemplary Encapsulated Digestive Enzyme PreparationSuitable for Pancreatic Enzymes: Packaging

In yet another further embodiment, the stability of the enzyme is due inpart to the encapsulation and in part to the trilaminar foil packaging.The following demonstrates the packaging process for the single dosesachets/pouches.

First, following manufacture the product is dispensed into clean, drumsdouble lined with food-grade polyethylene bags, and the drums aresealed. If specification criteria are met, the lot is then released fromquarantine, and the material is then shipped to a suitable packager forplacement into sachets for individual dosing to the patient.

For example a PD-73272 Printed Child Resistant (CR) Pouch consisting of26#C1S Paper/7.5#LDPE/0.0007″ Aluminum Foil/15# with a Surlyn liner isutilized for packaging. Preferably pre-printed film/foil, exteriorprinting will be with 1 color eye-mark on white background while in-lineprinting of lot number, expiration date and product code will also be in1 color, black. Overall sachet dimension are: W 2.50″×H 3.50″. Thesachet is sized to hold 900 mg of granules of Pancrelipaselipid-encapsulated drug product with a tolerance of ±10% into a unitdose pouch/sachet. The final product will have a protease activity ofnot less than 156 USP units/mg.

Example 9 An Exemplary Encapsulated Digestive Enzyme PreparationSuitable for Pancreatic Enzymes: Dissolution

The effect of the release of Pancreatase from lipid encapsulatedparticles with soy oil was studied using particles with varying levelsof lipid coating (expressed as % lipid coating per total particleweight. The coating level was varied from 10% to 30%. There was nosignificant effect of lipid coating in this range on the release ofpancreatase in an aqueous environment from the particles over a60-minute period. All formulations release over 80% of the enzyme withinthe first 30 minutes following the initiation of dissolution. Maximumrelease for the 90%, 80% and 70% particles was 85%, 88% and 83%respectively by 60 minutes.

Example 10 An Exemplary Enzyme Delivery System for Treatment of Autism

The choice of 70%-90% encapsulated pancreatic enzyme preparation (activeenzyme by weight) was selected on the basis of its release profile, assuitable for release of the enzyme in the proximal small intestineswhere protein digestion by the protease component will take place.

Soy oil was selected as the lipid coating, for its lack of proteincomponents, and corresponding lack of antigenic properties, to minimizeor eliminate the possibility of an allergic reaction to the lipidcoating in treated patients and children with autism.

The use of the 70-90% preparation increases pourability and flowproperties while decreases aerosolization, which permits use of a sachetor pouch delivery system.

The addition of the trilamminar foil housing insures that the sprinkleformulation will be stable, transportable, and will be delivered by asingle unit dose mechanism.

The low lipase formulation allow also for the safety by reducing thepotential for colonic strictures, and enhances the utilization of theprotease portion of the enzyme.

TABLE 5 Composition of LUMINENZ-AT encapsulated digestive enzymepreparation, 900 mg Sachets Compendial Ingredients Status FunctionsContent Pancreatic enzyme USP Active ingredient NLT 156 USP concentrate(porcine units/mg origin) Hydrogenated NF Lipid coating q.s. vegetableoil, Type I material (soybean oil)

The drug substance, pancreatic enzyme concentrate (porcine origin) ispurchased from an appropriate supplier. The properties of the pancreaticenzyme concentrate (pancreatin/pancrelipase) suitable for use in theproducts of this invention are described in the table below.

TABLE 6 Parameter USP Specification Protease (USP) NLT 25 USP Units/mgLipase (USP) NLT 2 USP Units/mg Amylase (USP) NLT 25 USP Units/mg Fat(USP) NMT 6.0%* Loss on Drying (USP) NMT 5.0% Escherichia coli (USP)Neg/10 g Salmonella species (USP) Neg/10 g *If less than 75 U/mgProtease, 6 U/mg Lipase or 75 U/mg Amylate, then specification is NMT3.0%Specifications for Hydrogenated Vegetable Oil (Soy Oil)Physical Appearance and Sensory Characteristics:Material provided in flake or powder form, free from foreign matter andobjectionable odor.

TABLE 7 Specification Analytical Procedure Chemical Parameter MeltingRange 67 to 69 C USP/NF <741> Class II Acid Value 0.4 Max. USP/NF <401>Iodine Value 5.0 Max. USP/NF <401>Method II Loss on drying 0.1% Max.USP/NF <731> Saponification Value 175-200 USP/NF <401> Heavy Metals0.001% Max. USP/NF <231> Organic Volatile Complies USP/NF <467>Method IVImpurities Residual Solvents Complies USP/NF <467> Unsaponifiable Matter0.8% Max. USP/NF <401> Microbial Parameters Total Aerobic Microbial 2000cfu/g max. USP/NF <61> Count Staphylococcus aureus Absent in 10 g USP/NF<61> Pseudomonas seruginosa Absent in 10 g USP/NF <61> SalmonellaSpecies Absent in 10 g USP/NF <61> Escherichia coli Absent in 10 gUSP/NF <61> Mold and yeast 200 cfu/g max. USP/NF <61>.

Chromatograms of working standard, diluent, mobile phase B, and placebodemonstrate no interference with the standard peak (see Chromatogram,FIG. 9). The analytical placebo and active tablet compositions are givenin Table 8.

TABLE 8 Composition of Analytical Placebo and Active Powder AnalyticalPlacebo Active Formulation Ingredients mg/sachet mg/sachet Pancreaticenzyme — 720.0 concentrate Lipid encapsulate 180.0 180.0 Total 180.0900.0

The method linearity was evaluated by analyzing several sample levels ofthe standard concentration in the presence of the placebo matrix. Theselevels were 50%, 70%, 100%, 130%, and 150%. Three injections of eachsample were used to calculate the average response (area/concentration)for that level. Then the relative standard deviation for the generatedresponse ratios was calculated along with the least-squares linearregression statistics for the average peak area vs. concentration (seeTables 9 and 10). A plot of the average peak area vs. concentration withthe linear regression line is given in FIG. 10.

TABLE 9 Linearity Data Average Standards Peak mg/ Peak Area (mAU s) AreaAverage mL % Injection 1 Injection 2 Injection 3 (mAU s) Response 0.10050 709.4 712.9 710.5 710.9 7109.3 0.140 70 1041.2 1040.0 1002.9 1028.07343.1 0.200 100 1529.0 1499.0 1523.1 1517.0 7585.2 0.260 130 1969.42010.3 1996.2 1992.0 7661.4 0.300 150 2336.2 2322.6 2350.6 2336.5 7788.2

TABLE 10 Linearity Results Parameter Criterion Result CorrelationCoefficient >0.997 0.999 y Intercept ±2.0% −112.9 RSD of Response Ratios<2.0% 3.6 Visual Linear yes Standard error of y intercept — 18.8 Slope —8139.1

Example 11 Biochemical Biomarkers, and Behavioral Core and Non-CoreSymptoms of Autism

The correlation between digestive enzyme deficiencies in autisticchildren was determined in children diagnosed with autism based onclinical (behavioral) symptoms. This correlation was also studied inchildren diagnosed with autism and a genetic co-morbidity. Following theinitial discovery that autistic children exhibited self-imposed proteindietary restrictions, studies were conducted which indicated thatabnormally low levels of fecal chymotrypsin (FCT) is useful as abiomarker for autism.

In addition, the number of autistic patients responding to pancreaticenzyme replacement was also determined, based on biomarker measurementsand clinical symptoms. Changes in the gastrointestinal system as well asa change in the core symptoms of autism were examined. The table belowprovides an overview of the studies conducted at multiple physicianbased sites.

TABLE 11 Study Number Total # of Subjects Autism Non-autism 1 9 9 2 2626 3 46 25 21 4 54 54 5 463 266 197 6 320 64 256 7 33 33 8 42 25 17 9 6868 10 225 225 11 175 175

Initial observations were based on observation of self-imposed dietaryrestriction by almost all children with autism. Multiple studies werethen conducted to evaluate the ability of autistic children to digestprotein. A study of the physiology of protein digestion led to anexamination of the gastrointestinal system's cascade of digestiveenzymes, especially those involved in protein degradation, such aschymotrypsin. As a measure of dysfunction, it was determined that fecalchymotrypsin (FCT) levels in children suffering from autism wereabnormally low.

Study 1

This initial study was an exploratory one to determine if a small cohortof children with autism indeed would have abnormally low levels (<9.0)of fecal chymotrypsin. (FCT). The results of study 001 is shown in FIG.10

All 9 children with autism evidenced an abnormally low FCT level ofbelow 7 Units/gram. (Normal≧9.0). This observation in a small set ofchildren led to further examination of the potential for a physiologicallink to autism heretofore undiscovered.

Study 2

Study 2 was undertaken to determine if a larger cohort of children (26children) with autism also experienced abnormally low FCT levels. Levelsof fecal elastase-1, another pancreatic digestive enzyme present at lowamounts in pancreatic insufficiency, was also determined. Again, thelevels of FCT were abnormally low in 25 of the 26 children, falling at 8U/g or below. One child had an FCT level of 9 U/g. On the other hand,all of the children had normal levels of fecal elastase-1.

Study 3

In Study 3, FCT levels were determined in 46 children aged 2 years to 14years of age, 25 with autism and 21 without autism, The datademonstrated that the children with autism had abnormally low FCT levelsand those children who did not have autism had normal FCT levels, of 12U/g or higher. The results are summarized in FIG. 12. The top line inFIG. 12 shows the FCT levels in subjects who did not have autism, whilethe bottom line shows the FCT levels in subjects who did have autism.

Study 4

In Study 4, 54 children diagnosed with autism and a co-morbid geneticdisorder were examined for FCT levels. The data showed that the childrenwith autism and a co-morbid genetic disorder tested normal for FCTlevel.

As autism is determined by behavioral assessment, it was hypothesizedthat autism due to, or present with a known genetic disorder may have adiffering physiology from others with autism alone, or not due to aknown genetic disorder. Some genetic disorders have typical symptoms,while others may be more variable and overlap with autisticsymptomology. This study examined children with autism who were alsodiagnosed with another known condition, to determine if FCT levels wereabnormally low in these children.

Table 12 below represents 54 children diagnosed with autism who also hada genetic co-morbidity

TABLE 12 Children Diagnosed with Autism who also Have a GeneticCo-Morbidity FCT Level Co- U/g Morbidity 1 12 Fragile X 2 22Hallermann-Streiff syndrome 3 25.2 Trisomy 21 4 15.8 tranlocation on 9 518 Beckwith-Wiedemann syndrome 6 26.6 Trisomy 21 7 39.2 Trisomy 18 816.6 Rubenstein-Tabi syndrome 9 25.4 Fragile X 10 20.6 Prader-Willisyndrome 11 14.6 Trisomy 21 12 25.6 Rett syndrome 13 21.4 Klippel-Feilsyndrome 14 20.6 Rett syndrome 15 24.8 Duchenne Muscular Dystrophy 1612.2 Tourette syndrome 17 14.8 In-utero stroke 18 30 Trisomy 21 19 18.8Fragile X 20 17.6 Juvenile RA 21 18.8 In-utero stroke 22 34 Trisomy6 2322.2 Duchenne Muscular Dystrophy 24 18.8 Juvenile Diabetes 25 28.4Diabetes Type I 26 13.8 Adrenoleukodystrophy 27 44 Wilson's disease 2819.6 In-utero stroke 29 7.4 Diabetes Type I 30 23.4 Prader-Willisyndrome 31 14.4 22q13 32 15.4 Tourette syndrome 33 17.6 Lissencephaly34 22.4 Neutrophil Immunodeficiency syndrome 35 18.4 Diabetes Type I 3632.2 Tourette syndrome 37 14.6 Tetrasomy 18p 38 31 Hyper IgE syndrome 3926.6 Angelman Syndrome 40 17.4 Diabetes Type I 41 12.6 Rett syndrome 4234 Fragile X 43 17.4 Marfan syndrome 44 21.2 Waardenburg syndrome 4521.8 glutathione synthetase deficiency 46 6.0 Diabetes Type I 47 26.6Rubinstein-Taybi 48 34 Angelman Syndrome 49 25.2 Klinefelter Syndrome 5021.4 Brain bleed at birth 51 16.8 Turner Syndrome 52 23.4 Hypothyroidism53 15.8 Diabetes Type I 54 7.8 Brain damage of prematurity

Only two of the 54 children diagnosed with both autism and a geneticco-morbidity had abnormally low levels of FCT. Those children had Type Idiabetes. 52 of the 54 children registered FCT levels in the normalrange.

This further supports that low FCT levels are present in childrendiagnosed with 5 autism in the absence of another known geneticmorbidity.

Study 5

In Study 5, FCT levels were determined for 463 children aged 2 years to8 years of age, 266 diagnosed with autism and 197 diagnosed withoutautism, in a multi-office physician-conducted study. The data showedthat the children with autism had abnormally low fecal chyotrypsinlevels and those children who did not have autism had normal levels offecal chymotrypsin.

The data is summarized in table 13 below.

TABLE 13 Mean Fecal Chymotrypsin Levels in Children with and withoutAutism Children with Children not N = 463 Autism with Autism Totalnumbers of children 266 197 Mean FCT (U/g) 4.4 23.2 Total Children withAbnormal 203 3 Levels of FCT % (p < 0.001) 76.34% 1.50% Total Childrenwith Normal 63 194 Levels of FCT (p < 0.01) % 23.68% 98.50%

This data further established that children diagnosed with autism who donot also have a known genetic co-morbidity have abnormally low levels ofFCT. FCT levels may therefore be useful in diagnosing children withautism, if the child does not also have a known genetic co-morbidity(unless the co-morbidity is Type I diabetes).

Chymotrypsin is a pancreatic enzyme. Chymotrysin is a serine proteaseand is unique in that it cleaves only essential amino acids during thedigestive process. Specifically, chymotrypsin cleaves the peptide bondon the carboxyl side of aromatic amino acids. A lack of proteindigestion as evidenced by abnormal FCT levels leaves the child with adearth of amino acids available for new protein synthesis. Withoutsufficient levels of essential amino acids, new proteins required forvarious bodily functions cannot be synthesized. For example, a shortageor lack of proteins involved in neurological processes may then giverise to symptoms of autism.

Study 6

In Study 6, FCT levels were determined for 320 children aged 2 years to18 years of age, 64 with autism, 64 with ADD. 64 with ADHD, 64 withknown genetic conditions, and 64 normals (no known conditions). The datashowed that the children with autism, ADD and ADHD exhibited abnormallylow levels of FCT compared to the children with known genetic conditionsand normal children. FCT data were gathered during a multi-physicianoffice trial of age-matched children with multiple conditions. FIG. 13depicts FCT levels in separate groups of children aged 6 years to 18years who have Autism, ADHD (Attention Deficit Hyperactivity Disorder),ADD (Attention Deficit Disorder), known genetic disorder also diagnosedwith autism, or no known condition (normals).

The two upper lines in FIG. 13 correspond to FCT levels in childrenwithout any known condition and children with known co-morbid conditions(genetic and others). The three bottom lines correspond to FCT levels inthe children with autism, ADD and ADHD.

The Autism, ADD, and ADHD children had significantly lower levels FCTthan those without any known condition, or those with a known geneticco-morbidity or traumatic condition (p<0.01).

Study 7

In Study 7, 33 children who were diagnosed with autism and abnormallylow FCT levels were enrolled in the study. The children were treatedwith one of two pancreatic/digestive enzyme supplements, or given notreatment. FCT levels were measured for each child at time 0, 30, 60, 90and 120 days.

Eleven (11) children were given a low therapeutic dose of ULTRASE® MT20(pancrelipase) Capsules (opened to sprinkle on food) (see below); 11children were given Viokase® (pancrelipase) powder for sprinkling onfood at a minimal dosing level of/teaspoon; 11 children just had theirfecal chymotrypsin levels measured. All children were age-matched andwithout a co-morbid neurological and/or genetic diagnosis.

Each ULTRASE Capsule was orally administered and contained 371 mg ofenteric-coated minitablets of porcine pancreatic concentrate contained:

Lipase 20,000 U.S.P. Units Amylase 65,000 U.S.P. Units Protease 65,000U.S.P. UnitsEach 0.7 g (¼ Teaspoonful) of Viokase Powder contained:Lipase, USP units 16,800Protease, USP units 70,000Amylase, USP units 70,000

FCT levels were monitored over 120 days to determine whether FCT levelschanged in response to treatment with either of the enzyme formulations,compared to the children who did not receive enzyme treatment. Theresults of the FCT levels, measured over a 120 day period are shown inTable 14 below.

TABLE 14 Mean Fecal Chymotrypsin Levels at Baseline, 30, 60, 90 and 120days Post administration of Multiple Pancreatic Enzyme ReplacementUltrase Viokase No treatment Mean FCT (units) 3.49 3.81 3.1 At BaselineMean FCT (units) 5.05 7.02 3.15 30 Days Mean FCT (units) 4.82 8.96 3.1860 Days Mean FCT (units) 4.91 13.73 3.25 90 Days Mean FCT (units) 5.3815.1 3.13 120 Days N = 33

The results are shown in the bar graph in FIG. 14. The top bar (verypale bar) for each time point shows the FCT level for the untreatedchildren. The middle bar shows the FCT level for children treated withViokase, and the bottom bar at each time point shows the FCT levelfollowing Ultrase treatment. The results in the table and graphed inFIG. 14 indicate that a significant change in FCT level was seen onlyfollowing administration of the Viokase powder enzyme formula, frombaseline at time 0 to 120 days. The greatest change was seen in thefirst 90 days. The changes in the first 90 days were significantcompared to the changes seen between 90 and 120 days. While the Ultrasegroup showed some change from baseline to 120, the change was notsignificant.

The lipases in Ultrase are very sensitive to pH changes and todegradation in acidic conditions, such as those found in the stomach.The enteric coating on Ultrase allows the enzymes to bypass the stomach.Ultrase has been shown to be useful for delivery of sufficient lipasesto treat adults with cystic fibrosis and chronic pancreatitis who sufferfrom pancreatic enzyme deficiency. However, the enteric coating onUltrase and other similar products apparently did not allow the proteaseportion of those compositions to be delivered in the proximal smallintestine, where it is needed for protein degradation. As demonstratedin the small pilot study, Ultrase did not allow for release of theprotease portion of the enzyme, specifically chymotrypsin, as determinedby FCT levels measured following administration of Ultrase. The FCTlevels in the Ultrase treated group were similar to those found in theNO TREATMENT group.

The optimum delivery timing and location for the protease portion of theenzyme is from the latter portion of the time the bolus of food is inthe stomach, through the time the digesting food spends in the proximalsmall intestine.

Study 8

In Study 8, 42 age-matched children, 25 with autism, and 17 withoutautism or other co-morbid condition, were examined using a stool testfor the presence of multiple pathogens as well as markers ofGastrointestinal dysfunction, including FCT levels. The children withautism had a larger number of stool pathogens present as well asabnormally low FCT levels.

This small pilot study was undertaken to examine the gastrointestinalflora of children with autism versus those without autism. Multiplemarkers of gastrointestinal health were examined to determine if thereis an abnormal gastrointestinal presentation in these children.

42 children aged matched 25 with autism and 17 without autism or otherco-morbid condition were screened using a stool test for the presence ofmultiple pathogens as well as markers of Gastrointestinal dysfunction.Other GI pathogens or stool markers known to those of skill in the artmay also be tested as a marker of GI dysfunction. Table 15 below showsthe incidence of presence of a GI pathogen or other stool marker.

TABLE 15 Incidence of the Presence of Pathogens and other Stool MarkersRepresenting Gastrointestinal Dysfunction % NOT % AUTISM TOTAL AUTISMTOTAL LOW FCT 25 100% 0 0% C. difficile antigen 15 60% 1 6% FecalElastase < 200 0 0% 0 0% H. pylori antigen 17 67% 0 0% E. Histolyticaantigen 8 32% 0 0% Giardia antigen 9 36% 1 6% Yeast overgrowth 4 16% 00% Cryptosporidium 9 36% 1 6% N = 25 N = 17

The presence of positive stool markers in the children with autism,including low levels of fecal chymotrypsin indicated additionalgastrointestinal problems in patients with autism.

Study 9

In Study 9, 68 children aged 3 years to 8 years of age, diagnosed withautism who presented with abnormally low FCT levels were administered acombination of pancreatic/digestive enzymes for 90 days. Resultsdemonstrated significant improvement in 5 out of 5 areas representingboth the core and non-core symptoms of autism.

Examination of the multiple areas of symptomology in the children withautism in this study included both gastrointestinal symptoms as well asthe core symptoms of autism. It is well documented in the literaturethat children with autism do not change over time, and that their levelof autism is static regardless of the age of the child. Further there isthought to be no maturation changes accompanying those with autism.

In this study, 68 children aged 3-8 diagnosed with autism who presentedwith abnormally low FCT levels were administered ¼ teaspoonful ofViokase, and a chewable papaya enzyme (Original Papaya Enzyme Brand) ateach meal for a period of 90 days.

Original Papaya Enzyme

Supplement Facts Serving Size: 3 Tablets Servings Per Container: 33Carbohydrates <1 g <1% Sugars <1 g Papain 45 mg ** Amylase 6 mg **Protease 6 mg ** Papaya Fruit (Carica 3 mg ** papaya) *Based on a 2,000calorie diet **Daily Values not established

The physician and the parent were asked to complete a rating scale foreach of the symptoms examined in the study. Each symptom was rated onthe scale below with (0) indicating that the child is able to performthe task, thereby demonstrating no impairment, to (10) representing thechild's complete inability to perform the task. With respect toundesirable behaviors such as hyperactivity or obsessive compulsivebehavior, a change from a lower score to a higher score indicates animprovement, because the child is demonstrating the undesirable behaviorless often. The rating scale was as follows:

10 Child experiences a 0% ability to perform this task

9 Child can perform this task 10% of the time

8 Child can perform this task 20% of the time

7 Child can perform this task 30% of the time

6 Child can perform this task 40% of the time

5 Child can perform this task 50% of the time

4 Child can perform this task 60% of the time

3 Child can perform this task 70% of the time

2 Child can perform this task 80% of the time

1 Child can perform this task 90% of the time

0 Child can perform this task 100% of the time

The average of the two scores taken at each interval: baseline and 90days. The scores obtained are shown in Table 16 below:

TABLE 16 Symptom Scores for Children with Autism Pre- andPost-Administration of Digestive Enzymes Sum of Total Patient ScoresMean Score Sum Total Mean Score Pre- Pre- Patient Scores 90 Days PostDigestive Digestive 90 Days Post Enzyme Enzyme Enzyme Enzyme Admin AdminHyperactivity 300 4.41 568 8.35 Obsessive 255 3.75 554 8.15 CompulsiveBehavior Eye Contact 552 8.12 206 3.03 Speech 553 8.13 223 3.28 Partial515 7.57 197 2.9 Toilet Training N = 68

CARS scores have been used to study core symptoms of autism. In study 9,measures of core and non-core symptoms of autism were obtained(hyperactivity, obsessive compulsive behavior, eye contact, speech,partial toilet training) While the diagnosis of autism was made strictlyon the basis of a behavioral assessment of the core symptoms of autism,the study indicates that other non-core symptoms such as a lack oftoilet training, will lead to significant morbidity in this population.The 5 parameters measured in this study indicated that the increase intoilet training, eye contact, and speech as well as the decrease inhyperactivity and obsessive compulsive behaviors are core and non-coresymptoms that were improved by treatment with digestive enzymes.

Study 10 and Study 11

In Studies 10 and 11, 225 children ages 2-4 years of age, and 171children 5-11 years of age each of whom presented with abnormally lowlevels of fecal chymotrypsin, were administered a combination ofpancreatic/digestive enzymes 3 times a day for a period of 150 days.Nine total measures of autistic symptomotology, both core and non-core,were obtained at baseline and over a period of 150 days. Significantchanges representing improvements in both core and non-core symptomswere seen across all age levels, with the greatest change taking placeover the first 90 days.

Each of these studies were conducted similar to the protocol in STUDY 9.The children were divided into age groups of 2-4 and 5-11. In thesestudies, 225 children aged 2-4 and 171 aged 5-11 previously diagnosedwith autism who presented with abnormally low fecal chymotrypsin levelswere administered ¼ teaspoonful of Viokase, and a chewable papaya enzyme(Original Papaya Enzyme Brand) at each meal for a period of 150 days.The same rating scale used in STUDY 9 was utilized in these two studies.Additionally levels of toilet training, hand flapping, play habits, andformed bowel movements were assessed. The % of the cohorts thatexperienced changes were calculated as well. This study was extended to150 days, with no significance seen between day 90 and day 150.

Table 17 below shows the measurements obtained for the percentage ofchildren in each group who exhibited the indicated trait or behavior,including hyperactivity, obsessive compulsive behavior, hand flapping,eye contact, speech, partial toilet training, full toilet training,formed bowel movement and playing well with others.

TABLE 17 PERCENT (%) WITH TRAIT OR SYMPTOM FOLLOWING ENZYME REPLACEMENTAged 2-4, N = 225 Aged 5-11, N = 171 Therapy Day Therapy Day Day Day DayDay Day Measure Day 0 60 150 0 60 150 Had some eye contact 4 61 88 14 5989 Had some speech 23 58 75 18 64 86 Were partially toilet trained 8 6175 11 47 72 Were fully toilet trained 4 30 45 16 16 20 Had formed bowel15 88 100 16 18 97 movement Experienced hyperactivity 85 38 19 98 51 33Plays well with others 12 38 60 36 43 71 Experienced hand flapping 81 4631 1 75 36 Experienced other OCD 90 73 32 91 58 22

In studies 9, 10, and 11, measurements of core and non-core symptoms ofautism were obtained. While the diagnosis of autism has been madestrictly as a result of a behavioral assessment of the core symptoms ofautism, other non-core symptoms lead to significant morbidity in thispopulation. The lack of toilet training and formed bowel movements, forexample, create a hardship for parents, and often lead to a lack ofsocial integration, further contributing to the core symptoms of autism.This additional isolation due to the non-core symptoms of autism furtherimpedes the child's ability to learn and to integrate socially. Thisdynamic is continually present in this population. This effect can be asignificant driver of the core symptoms of autism. This demonstratesthat these non-core symptoms may also be valuable as indicators ofautism.

Example 12 Enzyme Delivery System Used in the Treatment of Autism

Encapsulated digestive enzyme preparations according to this inventionare packaged in pouches containing 900 mg/pouch, and are administered toa patient in need thereof by sprinkling the contents of one pouch ontofood just before serving, administered three times per day.Determination of whether a patient is in need of administration oftreatment with digestive enzymes including encapsulated digestive enzymepreparations such as those of this invention can be made using any testor indicator that is useful as a marker of a digestive enzymedeficiency. This determination is made, for example, using FCT levels,behavioral symptoms (core or non-core symptoms of autism), or detectionof a mutation in a gene affecting the activity and/or expression ofdigestive enzymes, for example, a MET gene mutation.

Relevant symptoms of the patient's condition or disease are measuredbefore and following a period of treatment. The percentage of patientsexhibiting some eye contact, some speech, partial toilet training, fulltoilet training, formed bowel movements, and ability to play well withothers increases at 60 days, or earlier than 60 days, with a furtherincrease at 150 days. The changes observed upon treatment with thedigestive enzymes of this invention take place over a shorter timecourse, and/or result in greater improvement in each individual at anygiven time point and/or improvements in core and non-core symptoms in ahigher percentage of individuals treated. In addition, a correspondingincrease in the number of patients exhibiting a decrease inhyperactivity, hand flapping, or another OCD is observed at 60 days,with a further increase in the number of patients exhibiting a decreasein those behaviors at 150 days.

Other core symptoms of autism such as those measured in a CARS test arealso observed and shown to improve following treatment.

What is claimed is:
 1. A coated free-flowing particulate digestiveenzyme preparation, comprising particles that comprise: (a) a corecomprising digestive enzymes, wherein the digestive enzymes comprise aprotease, an amylase and a lipase; and (b) a coating comprising anemulsifiable lipid, wherein the coating coats the core and theemulsifiable lipid emulsifies upon exposure to a solvent; wherein thedigestive enzymes are present in an amount of from 77.5-82.5% by weightof the particles, and wherein the coating masks the taste and smell ofthe digestive enzymes.
 2. The coated free-flowing particulate digestiveenzyme preparation of claim 1, wherein the coated free-flowingparticulate digestive enzyme preparation provides a therapeuticallyeffective amount of the digestive enzymes to treat a subject sufferingfrom autism, Attention Deficit Disorder (ADD), or Attention DeficitHyperactivity Disorder (ADHD).
 3. The coated free-flowing particulatedigestive enzyme preparation of claim 1, wherein the core is from about105 μm to about 425 μm.
 4. The coated free-flowing particulate digestiveenzyme preparation of claim 1, wherein the core is sieved from about 40mesh to about 140 mesh.
 5. The coated free-flowing particulate digestiveenzyme preparation of claim 1, wherein the preparation isnon-aerosolizable.
 6. The coated free-flowing particulate digestiveenzyme preparation of claim 1, wherein the emulsifiable lipid isselected from the group consisting of hydrogenated soy oil, hydrogenatedcastor wax, and carnauba wax.
 7. The coated free-flowing particulatedigestive enzyme preparation of claim 1, wherein about 80% of thedigestive enzymes are released by 30 minutes in a dissolution testperformed at pH 6.0.
 8. The coated free-flowing particulate digestiveenzyme preparation of claim 1, wherein the coating consists essentiallyof the emulsifiable lipid that is one or more monoglycerides.
 9. Thecoated free-flowing particulate digestive enzyme preparation of claim 1,wherein the emulsifiable lipid comprises monoglycerides.
 10. The coatedfree-flowing particulate digestive enzyme preparation of claim 1,wherein at least 90% of the particles are sieved from about #40 to about#140 mesh in size.
 11. The coated free-flowing particulate digestiveenzyme preparation of claim 1, wherein less than 20%, or less than 15%,of the particles can be sieved through #100 mesh.
 12. The coatedfree-flowing particulate digestive enzyme preparation of claim 1,wherein the emulsifiable lipid comprises at least one hydrophilic groupand at least one hydrophobic group.
 13. The coated free-flowingparticulate digestive enzyme preparation of claim 12, wherein theemulsifiable lipid is capable of forming a hydrophilic and hydrophobicinterface.
 14. The coated free-flowing particulate digestive enzymepreparation of claim 13, wherein the interface is a micelle interface.15. The coated free-flowing particulate digestive enzyme preparation ofclaim 13, wherein the interface is a bilayer interface.
 16. The coatedfree-flowing particulate digestive enzyme preparation of claim 12,wherein the emulsifiable lipid is obtained from animal or vegetableorigins.
 17. The coated free-flowing particulate digestive enzymepreparation of claim 16, wherein the emulsifiable lipid is selected fromthe group consisting of palm kernel oil, soybean oil, cottonseed oil,canola oil, poultry fat, hydrogenated soy oil, hydrogenated castor wax,and carnauba wax.
 18. The coated free-flowing particulate digestiveenzyme preparation of claim 1, wherein the emulsifiable lipid ishydrogenated, saturated or partially saturated.
 19. The coatedfree-flowing particulate digestive enzyme preparation of claim 1,wherein the emulsifiable lipid consists of hydrogenated soy oil.
 20. Thecoated free-flowing particulate digestive enzyme preparation of claim 1,wherein the emulsifiable lipid is selected from the group consisting ofmonoglycerides, diglycerides, fatty acids, esters of fatty acids,phospholipids, salts thereof, and combinations thereof.
 21. The coatedfree-flowing particulate digestive enzyme preparation of claim 20,comprising the esters of fatty acids, wherein the esters of fatty acidsare selected from the group consisting of acetic acid esters of mono-and diglycerides, citric acid esters of mono- and diglycerides, lacticacid esters of mono- and diglycerides, polyglycerol esters of fattyacids, propylene glycol esters of fatty acids, and diacetyl tartaricacid esters of mono- and diglycerides.
 22. The coated free-flowingparticulate digestive enzyme preparation of claim 1, wherein theemulsifiable lipid is a food grade emulsifiable lipid.
 23. The coatedfree-flowing particulate digestive enzyme preparation of claim 22,wherein the food grade emulsifiable lipid comprises sorbitanmonostearates, sorbitan tristearates, or calcium stearoyl lactylates.24. The coated free-flowing particulate digestive enzyme preparation ofclaim 1, wherein the core is processed by a technique selected from thegroup consisting of encapsulation, compression, extrusion and molding.25. The coated free-flowing particulate digestive enzyme preparation ofclaim 1, wherein at least 75% of the particles are sieved from about #40to about #80 mesh in size.
 26. The coated free-flowing particulatedigestive enzyme preparation of claim 1, wherein the emulsifiable lipidcomprises hydrogenated soy oil.
 27. The coated free-flowing particulatedigestive enzyme preparation of claim 1, wherein the coating consistsessentially of the emulsifiable lipid that is hydrogenated soy oil. 28.The coated free-flowing particulate digestive enzyme preparation ofclaim 1, wherein the digestive enzymes have a protease activity of notless than 156 USP units/mg.
 29. The coated free-flowing particulatedigestive enzyme preparation of claim 1, wherein the core comprises thedigestive enzymes present in an amount of 80% by weight of theparticles.
 30. The coated free-flowing particulate digestive enzyme ofclaim 1, wherein at least 90% of the particles are from about 105 toabout 425 μm.
 31. The coated free-flowing particulate digestive enzymepreparation of claim 1, wherein less than 20% of the particles can besieved through about 150 μm mesh.
 32. The coated free-flowingparticulate digestive enzyme preparation of claim 1, wherein less than15% of the particles can be sieved through about 150 μm mesh.
 33. Thecoated free-flowing particulate digestive enzyme preparation of claim 1,wherein at least 75% of the particles are from about 180 to about 425μm.
 34. The coated free-flowing particulate digestive enzyme preparationof claim 1 or 27, wherein the digestive enzymes are a pancreatincomposition.
 35. A pharmaceutical composition comprising atherapeutically effective amount of a coated free-flowing particulatedigestive enzyme preparation, wherein the preparation comprisesparticles that comprise: a core comprising digestive enzymes, whereinthe digestive enzymes comprise a protease, an amylase and a lipase; and,a coating comprising an emulsifiable lipid, wherein the coating coatsthe core and the emulsifiable lipid emulsifies upon exposure to asolvent; wherein the digestive enzymes are present in an amount of from77.5-82.5% by weight of the particles, wherein the coating masks thetaste and smell of the digestive enzymes and wherein the therapeuticallyeffective amount of the coated free-flowing particulate digestive enzymepreparation is effective to treat a subject suffering from autism,Attention Deficit Disorder (ADD), or Attention Deficit HyperactivityDisorder (ADHD).
 36. The pharmaceutical composition of claim 35,provided in a sachet or a pouch.
 37. The pharmaceutical composition ofclaim 35, wherein at least 80% of the digestive enzymes are released by30 minutes in a dissolution test performed at pH 6.0.
 38. Thepharmaceutical composition of claim 35, wherein at least 90% of theparticles are sieved from about #40 to about #140 mesh in size.
 39. Thepharmaceutical composition of claim 35, wherein less than 20% of theparticles can be sieved through #100 mesh.
 40. The pharmaceuticalcomposition of claim 35, wherein at least 75% of the particles aresieved from about #40 to about #80 mesh in size.
 41. The pharmaceuticalcomposition of claim 35, wherein the emulsifiable lipid compriseshydrogenated soy oil.
 42. The pharmaceutical composition of claim 35,wherein the coating consists essentially of the emulsifiable lipid thatis hydrogenated soy oil.
 43. The pharmaceutical composition of claim 35,wherein the digestive enzymes have a protease activity of not less than156 USP units/mg.
 44. The pharmaceutical composition of claim 35,wherein the core comprises the digestive enzymes present in an amount of80% by weight of the particles.
 45. The pharmaceutical composition ofclaim 35, wherein at least 90% of the particles are from about 105 toabout 425 μm.
 46. The pharmaceutical composition of claim 35, whereinless than 20% of the particles can be sieved through about 150 μm mesh.47. The pharmaceutical composition of claim 35, wherein less than 15% ofthe particles can be sieved through about 150 μm mesh.
 48. Thepharmaceutical composition of claim 35, wherein at least 75% of theparticles are from about 180 to about 425 μm.
 49. The pharmaceuticalcomposition of claim 35 or 42, wherein the digestive enzymes are apancreatin composition.
 50. A pharmaceutical composition comprising adose of a coated free-flowing particulate digestive enzyme preparation,wherein the preparation comprises particles that comprise: a corecomprising digestive enzymes, wherein the digestive enzymes comprise aprotease, an amylase and a lipase; and a coating to provide forcontrolled release of the digestive enzymes, wherein the coatingcomprises an emulsifiable lipid, wherein the emulsifiable lipid is a soylipid; wherein the digestive enzymes are present in an amount of from77.5-82.5% by weight of the particles, wherein the coating masks thetaste and smell of the digestive enzymes, and wherein the digestiveenzymes have a protease activity of not less than 156 USP units/mg. 51.The pharmaceutical composition of claim 50, wherein the digestiveenzymes are present in an amount effective for treatment of a subjectsuffering from autism, ADD, or ADHD.
 52. The pharmaceutical compositionof claim 50, wherein the emulsifiable lipid is hydrogenated soy oil,hydrogenated castor wax, or carnauba wax.
 53. The pharmaceuticalcomposition of claim 50, wherein the dose is provided in a sachet orpouch.
 54. The pharmaceutical composition of claim 50, wherein at least90% of the particles are sieved from about #40 to about #140 mesh insize.
 55. The pharmaceutical composition of claim 50, wherein less than20% of the particles can be sieved through #100 mesh.
 56. Thepharmaceutical composition of claim 50, wherein at least 75% of theparticles are sieved from #40 to about #80 mesh in size.
 57. Thepharmaceutical composition of claim 50, wherein the coating consistsessentially of the emulsifiable lipid that is hydrogenated soy oil. 58.The pharmaceutical composition of claim 50, wherein the core comprisesthe digestive enzymes present in an amount of 80% by weight of theparticles.
 59. The pharmaceutical composition of claim 50, wherein atleast 90% of the particles are from about 105 to about 425 μm.
 60. Thepharmaceutical composition of claim 50, wherein less than 20% of theparticles can be sieved through about 150 μm mesh.
 61. Thepharmaceutical composition of claim 50, wherein less than 15% of theparticles can be sieved through about 150 μm mesh.
 62. Thepharmaceutical composition of claim 50, wherein at least 75% of theparticles are from about 180 to about 425 μm.
 63. The pharmaceuticalcomposition of claim 50 or 57, wherein the digestive enzymes are apancreatin composition.
 64. The pharmaceutical composition of claim 50,wherein the dose contains about 900 mg of the coated free-flowingparticulate digestive enzyme preparation.
 65. The pharmaceuticalcomposition of claim 50, wherein the coating is generally uniform.